CN108998538A - A kind of spot Ji SNP marker and its screening technique and application - Google Patents
A kind of spot Ji SNP marker and its screening technique and application Download PDFInfo
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Abstract
The invention discloses a kind of spot Ji SNP marker and its screening technique and application, the screening of spot Ji SNP marker includes extracting genomic DNA, RAD library construction and high-flux sequence, original sequence data filtering, the detection of SNP informative site and screening；Cyclic DNA and chitin are added during extracting genomic DNA, SNP marker screening accuracy can be effectively improved and obtains efficiency；The spot Ji SNP marker obtained with this method, can provide important basis for the correlative study of the analyses such as spot Ji population genetic diversity level, Genetic Constitution of Population.The utility model has the advantages that the method that the present invention obtains spot Ji SNP marker site has many advantages, such as high-throughput, low cost, SNP marker screening accuracy is high, it is high-efficient to obtain.
Description
Technical field
The present invention relates to technical field of molecular biology, more particularly, to a kind of spot Ji SNP marker and its screening technique and
Using.
Technical background
Spot Ji (Konosirus punctatus) is subordinate to Clupeiformes (Clupeiformes), clupeidae (Clupeidae), Ji
Subfamily (Dorosomatinae), spot Ji belong to (Konosirus), be coastal water warm pelagic fishes, be distributed widely in China,
Korea, South Korea and Japan are coastal, and in China, there is output at Bohai and Yellow Seas, the East Sea, the South Sea, are China, Japan and the Korea peninsula
The important fished species of inshore fishing.Spot Ji hands over the sediment resuspension and water-bottom interface substance of coastal ecosystems
Change important in inhibiting；It is the prey of a variety of high nutrition grade fish again simultaneously, plays and hold in marine food web frame
On open under pivotal player.Meanwhile because of its dressed fish height, fine and tender taste, delicious flavour, be by favor high-quality aquatic products it
One, people are to the demand of spot Ji also in steady-state growth in recent years.
Since Global climate change, environmental pollution and habitat destruction etc. influence in recent years, so that spot Ji wild population number
Amount greatly reduces.In addition the fishing pressure of lasting high intensity, China spot Ji population has already appeared miniaturization, becomes younger, sex premature
Etc. trend, if things go on like this will certainly genetic diversity to spot Ji and germ plasm resource cause considerable damage, generate difficult to the appraisal
Loss.Due to the crucial indicator species that it is not only important fishery fished species or marine ecosystems change, followed in substance
Irreplaceable role is played in ring and energy flow, the decline of spot Ji resource not only results in its own fishery production substantially
It reduces, but also the energy cycle of matter web frame of entire coastal ecosystems can be destroyed.Therefore, reinforce the pipe to spot Ji resource
It manages and protects and is very urgent.The pedigree geographical configuration and population dynamic history of spot Ji are studied, can be the reasonable development of spot Ji resource
Important scientific guidance is provided using with administrative protection, is all had to the sound development of the supplement and the ecosystem of decline germ plasm resource
There is important scientific meaning.
Single nucleotide polymorphism (Single nucleotide polymorphism, SNP) refers to that certain biology is different
It is polymorphic caused by the polymorphism that single nucleotide acid point mutation generates in individual DNA sequence dna, including displacement, transversion, insertion or missing
Phenomenon.SNP is the third generation New molecular marker technology after RFLP, SSR proposed by American scholar Lander in 1996,
" SNPs and complex genome " international conference is all held every year within 1998~2002 years to apply to inquire into SNPs in complex genome,
With the continuous development of the technologies such as PCR, parting detection, DNA chip, SNP tends to high-throughput, automation.In recent years, molecular labeling
Technology is increasingly becoming the important means of research aquatic livestock science of heredity, and for SNP as third generation genetic marker, bit number of points are more, close
Degree is wide, genetic diversity is high and the degree of automation is strong, can show the heredity letter that other molecular marking techniques are difficult to detect
Polymorphism is ceased, so, SNP marker technology is of great significance for aquatic livestock genetics research.
Highdensity SNP site is obtained based on advanced simplified genomic sequencing technique, can be found out from full-length genome level
The important Population Genetics parameters such as level of genetic diversity, the Genetic Constitution of Population of coastal area of china Huang crucian carp population, can overcome biography
Marine organisms population genetic is assessed dependent on single or a small number of gene locis in system method and evolutionary mechanism bring is uncertain
Property and the not high problem of resolution ratio, will be managed and Reasonable Protection provide background knowledge for the effective of yellow crucian carp, and facilitate in promotion
Marine fishes group of state genomics is studied to more Shenzhen Development Bank.
There are mainly two types of the methods for obtaining gene information data at present: Genotyping and gene sequencing.Genotyping is main
It is the base sequence design probe using the site known single nucleotide polymorphism (SNP) flank, and probe is fixed on gene
On chip.It is found out when the probe sequence Complementary hybridization on the DNA and genetic chip of sample to be tested and by scanning fluorescence signal
Hybridization, can identify the genotype of these probe sites (SNP site) on sample DNA.And gene sequencing is carried out to target dna
Base sequence measurement, and carry out correlation analysis.
The prior art such as Authorization Notice No. is the Chinese invention patent of 105238859 B of CN, and it is complete to disclose a kind of acquisition chicken
The method in genome high-density SNP marker site, comprising the following steps: (1) the prediction double digestion chicken gene of EcoRI and MseI
Group endonuclease bamhi distribution situation obtained；(2) according to the endonuclease bamhi characteristic distributions of EcoRI and MseI design universal joint,
Bar code connector and PCR amplification primer；(3) it constructs and simplifies gene order-checking library；(4) using step (3) building library into
Machine is sequenced on row；(5) SNP marker site is obtained according to sequencing result；The invention obtains the cost in each SNP marker site than passing
System chip technology reduces an order of magnitude, and method and technology is stablized, and repeatability is high.However invention sequencing program is more complex, mesh
It is low to mark DNA acquisition purity.
Summary of the invention
The purpose of the present invention is to provide a kind of spot Ji SNP marker screening techniques, using polymerase chain reaction and series connection
RAD label sequencing technology screening spot Ji SNP marker, has high-throughput, low cost, and SNP marker screens accuracy height, obtains efficiency
The advantages that high.
It, can be from full base the present invention also aims to obtain highdensity SNP site using spot Ji SNP marker screening technique
Because group level finds out the important Population Genetics ginseng such as level of genetic diversity, Genetic Constitution of Population of coastal area of china spot Ji population
Number can overcome in conventional method and assess marine organisms population genetic and evolutionary mechanism dependent on single or a small number of gene locis
Bring uncertainty and the not high problem of resolution ratio will provide background knowledge for effective management of spot Ji and Reasonable Protection, and
Help that Chinese Sea fish group genomics is pushed to study to more Shenzhen Development Bank.
The present invention in view of the above technology in the problem of mentioning, the technical solution taken are as follows:
A kind of spot Ji SNP marker screening technique, comprising the following steps:
Extract genomic DNA: extraction solution includes extracting solution I and extracting solution II；Wherein extracting solution I include buffer salt,
The aqueous solution of nonionic surfactant and neutral salt, pH value 7.5-8.5；Extracting solution II include buffer salt and sun from
Sub- surfactant, pH value 7.8-8.2；
Specific steps are as follows: spot Ji muscle or isozyme sample are shredded, extracting solution I is placed in, cyclic DNA and crust is added
Extracting solution II is added after collecting supernatant in matter, freeze thawing 2-3 times, centrifugation, and after being mixed by inversion, centrifugation generates white precipitate, and washing is heavy
It forms sediment to obtain DNA sample, for use；
RAD library construction and high-flux sequence: include the following steps:
A1. digestion: carrying out endonuclease reaction to N number of genomic DNA using selected restriction endonuclease respectively, obtain N parts of endonuclease bamhis,
N is the integer greater than 2；
A2. connector connects: being separately connected connector to N parts of endonuclease bamhis, i.e. design N butt joint combination, obtains N parts of connections and produce
The connector of object, the connection of every part of endonuclease bamhi both ends is designed with the restriction enzyme site of SapI enzyme and for realizing the concatenated feature of label
The universal sequence that sequence and amplimer combine, the series sequence of N group endonuclease bamhi is determined according to added connector；
A3. connection product expands: by the obtained N part connection product of step A2 be utilized respectively different biotin primer with
General primer combination carries out PCR amplification, and enrichment is connected with the endonuclease bamhi of connector, gel extraction PCR product, using same side
Method expands 4-8 circulation, and the PCR product of N parts of enrichments is obtained after amplification；The PCR product mixed in equal amounts that N parts are enriched with, and carry out
Purifying；
A4. it connects tag library: using the N part PCR product progress digestion of SapI enzyme to mixing and after purification, having cut off enzyme
It is sliced section both ends general connector and primer sequence, retain the characteristic sequence having on connector and forms end viscosity protrusion, N
Part PCR product form can direct concatenated label, according to the characteristic sequence complementary pairing on connector, make N parts of tag libraries by
According to being sequentially sequentially connected in series, must connect long label；
A5. long label of connecting is enriched with: the long label that will connect utilizes primer to carry out PCR amplification after gel-purified, introduces
Barcode building series connection tag library；
A6. library is sequenced: series connection tag library is sequenced using Illunima microarray dataset；
Original sequence data filtering: it filters out in raw sequencing data comprising band Adaptor information, low quality base and not
The reads pair for the base measured；These information included in raw sequencing data can cause very subsequent information analysis
Big interference gets rid of these interference informations before analysis, and obtained valid data provide for the Accurate Analysis of follow-up data
Guarantee；
The detection of SNP informative site and screening: resulting reads pair will be filtered and pass through BWA software and the spot Ji delivered
Draft genome is compared, and then uses the bayesian algorithm model in SAMTOOLS 0.1.19 to be with maximum reads depth
1000 parameter setting carries out the detection of SNP, filters to obtain spot Ji SNP marker site.
Preferably, cyclic DNA is the bacteria plasmid DNA of high-purity；Different size of ring-type can be selected as needed
DNA, as long as added cyclic DNA and constructed target dna library can efficiently separate；The spy of cyclic DNA and chitin
It is different to exist, it on the one hand can promote hydrone in spot Ji histocyte that aquation occurs in conjunction with nonionic surfactant,
It is destroyed to which the hydration shell to spot Ji histocyte protein surface layer is constituted, causes protein to be easy to assemble to form precipitating, simultaneously
Since it has the dielectric constant that can reduce extracting solution I, increase is extracted in spot Ji histocyte between opposite charges group
Attraction, promote the aggregation and precipitating of protein molecule, effectively increase the removal efficiency of spot Ji histocyte protein, mention
The high purity for being extracted tissue DNA；On the other hand, when being mixed with a large amount of microorganisms (or other heterogeneity biologicals) genome in sample
Pollution, cyclic DNA and chitin have effects that its structure is polluted and destroyed in identification, so that extracting target dna can be interference-free
Ground progress is subsequent to build library and sequencing, thus SNP marker screening accuracy is high, it is high-efficient to obtain.
Preferably, buffer salt in Tris-HCl, sodium phosphate, disodium hydrogen phosphate and sodium dihydrogen phosphate at least one
Kind, concentration 100-200mM.
Preferably, nonionic surfactant is the fatty alcohol ether ammonium sulfate or fatty alcohol-ether sodium sulfate of C12~C18.
Preferably, cationic surfactant is cationic polyacrylamide, concentration 0.1-5ppm.
Preferably, neutral salt is sodium chloride or potassium chloride, concentration 100-200mM.
Preferably, the temperature of freezing is -196 to -20 DEG C during extracting genomic DNA, the temperature of thawing is 40-
70℃。
Preferably, in A1 step restriction endonuclease be IIB type restriction enzyme, methyl modification dependent form restriction endonuclease in one
Kind is several.
Preferably, the design of A2 step center tap, by taking 3 butt joints as an example, three butt joints combination is respectively S1a and S1b,
S2a and S2b, S3a and S3b, each connector are made of two nucleotide fragments, the digestion of SapI in the sequence of connector S1a and S3b
Site devises the mutation of a base, cannot be digested, when using SapI enzyme to the PCR product digestions of three kinds of hybrid tags,
Two end connector S2a and S2b of digestion label and the connector and primer universal sequence of the side S1b and S3a can be removed by SapI digestion, be made
It is prominent that three base characteristic sequences that three kinds of label segment two sides have form end viscosity, according to the complementary pairing of characteristic sequence,
Realize that three kinds of labels head and the tail are sequentially connected in series to form series connection label, and the universal sequence of S1a and S3b tip side is still on label of connecting
Retain, the amplification enrichment for next step series connection label provides the binding site of primer.
Further preferably, two nucleotide fragments of S1a are constituted, sequence is respectively SEQ ID NO:1 and SEQ ID
NO:2；Two nucleotide fragments of S1b are constituted, sequence is respectively SEQ ID NO:3 and SEQ ID NO:4；Constitute the two of S2a
A nucleotide fragments, sequence are respectively SEQ ID NO:5 and SEQ ID NO:6；Two nucleotide fragments of S2b are constituted,
Sequence is respectively SEQ ID NO:7 and SEQ ID NO:8；Two nucleotide fragments of S3a are constituted, sequence is respectively SEQ ID
NO:9 and SEQ ID NO:10；Two nucleotide fragments of S3b are constituted, sequence is respectively SEQ ID NO:11 and SEQ ID
NO:12。
A kind of application of spot Ji SNP marker, the spot Ji SNP marker site using acquisition are spot Ji population genetic diversity
The correlative study of the analyses such as level, Genetic Constitution of Population provides important basis.
Compared with the prior art, the advantages of the present invention are as follows: 1) present invention can be achieved construct multiple labels series connection sequencing
Library so that label sequencing cost substantially reduces, realize it is high-throughput to full-length genome range genetic marker, at low cost screening and
Detection；2) cyclic DNA and chitin are added when extracting target gene group DNA by the present invention, can be improved the purity for obtaining DNA,
And pollutant and destroy its structure in identification target dna, thus be conducive to extract target dna can uninterruptedly carry out it is subsequent
Build library and sequencing；3) by simplifying genomic sequencing technique, genome-wide screening is carried out to spot Ji, obtains highdensity SNP
Site, and the analysis such as level of genetic diversity, Genetic Constitution of Population is carried out to spot Ji population using the site screened.
Specific embodiment
The present invention program is described further below by embodiment:
Embodiment 1:
A kind of spot Ji SNP marker screening technique, comprising the following steps:
It extracts genomic DNA: spot Ji muscle or isozyme sample being shredded, being placed in extracting solution I:70ml concentration is
The Tris-HCl and disodium hydrogen phosphate of 150mM, 0.5g fatty alcohol ether ammonium sulfate, 30ml concentration are the sodium chloride solution of 130mM；Add
Entering 0.5 μ g Plasmid DNA and 0.1 μ g chitin, freeze thawing 2-3 times, the temperature of freezing is -166 DEG C, and the temperature of thawing is 50 DEG C, from
The heart is collected and Tris-HCl that extracting solution II:10ml concentration is 100mM is added after supernatant and disodium hydrogen phosphate and 30ml concentration are
The cationic polyacrylamide of 1.6ppm, after being mixed by inversion, centrifugation generates white precipitate, and washing precipitates to obtain DNA sample, for use；
On the one hand the special presence of cyclic DNA and chitin can promote hydrone and non-ionic surface active in spot Ji histocyte
Agent combines, and aquation occurs, and destroys to constitute to the hydration shell on spot Ji histocyte protein surface layer, causes protein easy
Precipitating is formed in aggregation, simultaneously because it has the dielectric constant that can reduce extracting solution I, increase is extracted spot Ji histocyte
Attraction between middle opposite charges group promotes the aggregation and precipitating of protein molecule, and it is thin to effectively increase spot Ji tissue
The removal efficiency of born of the same parents' protein improves the purity for being extracted tissue DNA；On the other hand, when be mixed in sample a large amount of microorganisms (or
Other heterogeneity biologicals) genome pollution, cyclic DNA and chitin have effects that identification pollutes and destroys its structure, so that mentioning
Take target dna can uninterruptedly carry out it is subsequent build library and sequencing, thus SNP marker screening accuracy is high, it is high-efficient to obtain；
RAD library construction and high-flux sequence:
1) two kinds of IIB type restriction enzyme (BsaXI, BcgI) digestion genomic DNAs are selected, three kinds of different types are obtained
Digestion products；Digestion system is 15 μ L, includes 200ng genomic DNA, the restriction endonuclease (NEB) of 1U, 1 × cutsmart, digestion
Reaction temperature is 37 DEG C, keeps the temperature 45min；
2) different splice combinations are separately connected to three parts of digestion products, as shown in table 1, obtain three parts of connection products；Even
Connecing reaction system is 20 μ L, includes 10 μ L digestion products, 200U T4DNA ligase (NEB), 1 × T4Ligase Buffer, 4 μ
Mol/L Slx-Sa, 4 μm of ol/L Slx-Sb, 10mmol/L adenosine triphosphate atps, connection reaction temperature are 16 DEG C, connect 1h；
The splice combinations that three parts of digestion products are connected in 1 embodiment 1 of table
| Label position | Slx-Sa | Slx-Sb |
| Label 1 (BsaXI) | S1a | S1b |
| Label 2 (BcgI) | S2a | S2b |
| Label 3 (BsaXI) | S3a | S3b |
Wherein constitute two nucleotide sequences of S1a are as follows:
5 '-ACACTCTTACCCGACGCTGTTCCGATCTNNN-3 ' (SEQ ID NO:1) and 5 '-AGATCGGAACAGC-
3'(SEQ ID NO:2)；
The nucleotide sequence of S1b are as follows:
5 '-GTGACTGCAGGCGTGTGCTCTTCACGANNN-3 ' (SEQ ID NO:3) and 5 '-TCGTGAAGAGCAC-
3'(SEQ ID NO:4)；
The nucleotide sequence of S2a are as follows:
5 '-ACACTCTAACACGACGCTCTTCAGTCNNN-3 ' (SEQ ID NO:5) and 5 '-GACTGAAGAGCGT-3 '
(SEQ ID NO:6)；
The nucleotide sequence of S2b are as follows:
5 '-GTGACTGGAGTTCCGACGTCTTCACAGNNN-3 ' (SEQ ID NO:7) and 5 '-CTGTGAAGAGCAC-
3'(SEQ ID NO:8)；
The nucleotide sequence of S3a are as follows:
5 '-ACACTCTTTGCCTACACGCTTCACTGNNN-3 ' (SEQ ID NO:9) and 5 '-CAGTGAAGAGCGT-3 '
(SEQ ID NO:10)；
The nucleotide sequence of S3b are as follows:
5 '-GTGACTGGAGTCCAGTGCTGTCCGATCTNNN-3 ' (SEQ ID NO:11) and 5 '-
AGATCGGAACAGC-3'(SEQ ID NO:12)；
NNN be annex base, N represent four kinds of bases A, G, C, T any one, BsaXI digestion postgenome generate mark
There are three the cohesive ends of base random combine for label tape, therefore connector design herein has 3 merger bases to enable connector
It is enough to be connect with the label in genome by cohesive end；
3) PCR amplification is carried out to the three parts of connection products obtained in step 2), is enriched with endonuclease bamhi, obtained three parts of PCR and produce
Object；Three parts of PCR products, 8% non-denaturing polyacrylamide fine jade detected through gel electrophoresis, amplified production size is about 100bp, cuts glue
Recycle three parts of PCR products；Three parts of PCR products of recycling are subjected to amplification enrichment again respectively, what amplification 7 recycled finally
PCR product；Three parts of final PCR products are mixed in equal volume, are purified using the MinElutePCRkit of Qiagen company,
Obtain portion PCR purified product；
4) digestion is carried out to mixing PCR product using SapI enzyme, makes endonuclease bamhi formation can concatenated tag library；It utilizes
8% non-denaturing polyacrylamide fine jade detected through gel electrophoresis series connection label product, connection product size is about 244bp, gel extraction
Connection product；
5) series connection label product utilization primer further expands, and introduces required for the sequencing of Barcode and Illunima platform
Universal sequence；PCR product is detected with 8% native polyacrylamide gel electrophoresis, and amplified production size is about 299bp, benefit
With the MinElute PCR product purification kit recovery purifying PCR product of Qiagen company.It is surveyed using Illunima Hiseq
The sequencing of sequence platform；
Original sequence data filtering: DNA library is sequenced by IlluminaHiSeq4000 platform, is sequenced
Primitive sequencer sequence is initial data, and belt lacing (Adaptor) information, low quality base and not can be included in raw sequencing data
The reads pair for the base (being indicated with N) measured, however these information can cause very big interference to subsequent information analysis,
It needs to get rid of these interference informations before analysis, finally obtained data are valid data, in addition, obtaining valid data
Afterwards, it needs further to remove since PCR replicates caused repetition reads (duplication) and to the quality score of data
The distribution of (Q20, Q30) and G/C content is assessed, to guarantee the requirement of subsequent data analysis, the standard of filtering are as follows: filter out and contain
There is the reads pair of joint sequence；Containing for N base in read (N indicating the base information that sequencing can not determine) is sequenced when single-ended
When amount is more than 10%, this is removed to double end reads；When the low quality (score≤5 phred) contained in single-ended sequencing read
When base number is more than the 50% of this read length, this is removed to double end reads pair；It filters out since PCR amplification generates
Repetitive sequence；
The detection of SNP informative site and screening: resulting reads pair will be filtered and pass through BWA software and the spot Ji delivered
Draft genome is compared, and then uses the bayesian algorithm model in SAMTOOLS 0.1.19 to be with maximum reads depth
1000 parameter setting carries out the detection of SNP, is finally filtered to the SNP site detected according to standard below: SNP's
Mass value is not less than 30 (Q30)；The reads of SNP supports number to be not less than 10；The individual coverage of SNP is not less than 80%；Minimum etc.
Position gene frequency (MAF) is not less than 5%；Only retain biallelic marker；Each group's heterozygosity HO≤0.5；The coefficient of inbreeding-
0.3≤FIS≤0.3；After filtering by parameters such as depth, coverages, 49,694 spot Ji SNP marker sites are obtained.
These spot Ji SNP marker sites obtained can be spot Ji population genetic diversity level, Genetic Constitution of Population etc.
The correlative study of analysis provides important basis.
Embodiment 2:
It extracts genomic DNA and is added without cyclic DNA and chitin in the process, rest part and embodiment 1 are completely the same；Most
Whole filtering screening obtains 28730 spot Ji SNPs, this illustrates that cyclic DNA and crust mass-energy, which is added, to be effectively improved and extract the complete of DNA
Degree help to obtain spot Ji full-length genome information, improves SNP marker screening accuracy and acquisition rate.
Embodiment 3:
During RAD library construction and high-flux sequence, chitin fluid, crust are added during PCR amplification system
The mass percent of matter is 0.16%, and solvent is l-amino acid ethyl ester；Rest part and embodiment 1 are completely the same；Crust mass flow
The special presence of body has extremely strong adsorption capacity to DNA, can be improved PCR success rate, while chitin fluid is by many micro-
Spherula composition, can wrap up reaction system, complete pcr amplification reaction in separate space small one by one, to improve
The reaction susceptibility of PCR amplification keeps reaction more abundant, improves the PCR amplification effect of minim DNA；In addition, chitin fluid energy
The boiling point for improving reaction system is not easy to evaporate system buffer, the dosage of reagent during PCR amplification is reduced, to reduce
Experimental cost.
Routine operation in operation of the present invention step is well known to those skilled in the art, herein without repeating.
Technical solution of the present invention is described in detail in embodiment described above, it should be understood that the above is only
For specific embodiments of the present invention, it is not intended to restrict the invention, all any modifications made in spirit of the invention,
Supplement or similar fashion substitution etc., should all be included in the protection scope of the present invention.
Sequence table
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Claims (10)
1. a kind of spot Ji SNP marker screening technique, including extract genomic DNA, RAD library construction and high-flux sequence, original
Sequence data filtering, the detection of SNP informative site and screening, it is characterised in that: the step of the extraction genomic DNA are as follows: by spot
Ji muscle or isozyme sample shred, and are placed in extracting solution I, and cyclic DNA and chitin is added, and freeze thawing 2-3 times is centrifuged, and collect
Extracting solution II is added after supernatant, after being mixed by inversion, centrifugation generates white precipitate, and washing precipitates to obtain DNA sample, for use.
2. a kind of spot Ji SNP marker screening technique according to claim 1, it is characterised in that: the cyclic DNA is high-purity
The bacteria plasmid DNA of degree.
3. a kind of spot Ji SNP marker screening technique according to claim 1, it is characterised in that: the extracting solution I includes slow
Rush the aqueous solution of salt, nonionic surfactant and neutral salt, pH value 7.5-8.5；Extracting solution II include buffer salt and
Cationic surfactant, pH value 7.8-8.2.
4. a kind of spot Ji SNP marker screening technique according to claim 1, it is characterised in that: the RAD library construction with
High-flux sequence includes the following steps:
A1. digestion: using selected restriction endonuclease endonuclease reaction is carried out to N number of genomic DNA respectively, obtains N parts of endonuclease bamhis, N is
Integer greater than 2；
A2. connector connects: connector is separately connected to N parts of endonuclease bamhis, i.e. design N butt joint combination, obtains N parts of connection products,
The connector of every part of endonuclease bamhi both ends connection is designed with the restriction enzyme site of SapI enzyme and for realizing the concatenated feature sequence of label
The universal sequence that column and amplimer combine, the series sequence of N group endonuclease bamhi is determined according to added connector；
A3. connection product expands: the obtained N part connection product of step A2 is utilized respectively different biotin primer and commonly
Primer combination carries out PCR amplification, and enrichment is connected with the endonuclease bamhi of connector, and gel extraction PCR product is expanded using same method
Increase 4-8 circulation, the PCR product of N parts of enrichments is obtained after amplification；The PCR product mixed in equal amounts that N parts are enriched with, and purified；
A4. it connects tag library: using the N part PCR product progress digestion of SapI enzyme to mixing and after purification, having cut off digestion piece
Section both ends general connector and primer sequence retain the characteristic sequence having on connector and form end viscosity protrusion, and N parts
PCR product form can direct concatenated label, according to the characteristic sequence complementary pairing on connector, make N parts of tag libraries according to
Sequence is sequentially connected in series, and must connect long label；
A5. long label of connecting is enriched with: the long label that will connect utilizes primer to carry out PCR amplification after gel-purified, introduces barcode
Building series connection tag library；
A6. library is sequenced: series connection tag library is sequenced using Illunima microarray dataset.
5. a kind of spot Ji SNP marker screening technique according to claim 4, it is characterised in that: inscribe in the A1 step
Enzyme is one or more of IIB type restriction enzyme, methyl modification dependent form restriction endonuclease.
6. a kind of spot Ji SNP marker screening technique according to claim 4, it is characterised in that: the A2 step center tap
Design, by taking 3 butt joints as an example, three butt joints combination is respectively S1a and S1b, S2a and S2b, S3a and S3b, each connector by
Two nucleotide fragments form, and the restriction enzyme site of SapI devises the mutation of a base in the sequence of connector S1a and S3b, no
Can be digested, when using SapI enzyme to the PCR product digestions of three kinds of hybrid tags, two end connector S2a and S2b of digestion label with
And the connector and primer universal sequence of the side S1b and S3a can be removed by SapI digestion, three bases for having three kinds of label segment two sides
Characteristic sequence forms end viscosity and protrudes, and according to the complementary pairing of characteristic sequence, realizes that three kinds of labels head and the tail are sequentially connected in series to be formed
Series connection label, and the universal sequence of S1a and S3b tip side still retains on label of connecting, for the amplification for label of connecting in next step
Enrichment provides the binding site of primer.
7. a kind of spot Ji SNP marker screening technique according to claim 6, it is characterised in that: two for constituting S1a
Nucleotide fragments, sequence are respectively SEQ ID NO:1 and SEQ ID NO:2；Constitute two nucleotide fragments of S1b, sequence
Column are respectively SEQ ID NO:3 and SEQ ID NO:4；Two nucleotide fragments of S2a are constituted, sequence is respectively SEQ ID
NO:5 and SEQ ID NO:6；Two nucleotide fragments of S2b are constituted, sequence is respectively SEQ ID NO:7 and SEQ ID NO:
8；Two nucleotide fragments of S3a are constituted, sequence is respectively SEQ ID NO:9 and SEQ ID NO:10；Constitute two of S3b
Nucleotide fragments, sequence are respectively SEQ ID NO:11 and SEQ ID NO:12.
8. a kind of spot Ji SNP marker screening technique according to claim 1, it is characterised in that: the original sequence data
Filtration step are as follows: filtering out includes with Adaptor information, low quality base and the base that do not measure in raw sequencing data
reads pair。
9. a kind of spot Ji SNP marker screening technique according to claim 1, it is characterised in that: the SNP informative site inspection
Survey and screening step are as follows: resulting reads pair will be filtered and carried out by BWA software with the spot Ji draft genome delivered
It compares, then uses the parameter setting that the bayesian algorithm model in SAMTOOLS 0.1.19 is 1000 with maximum reads depth
The detection for carrying out SNP, filters to obtain spot Ji SNP marker site.
10. a kind of described in any item applications of spot Ji SNP marker of claim 1-9, it is characterised in that: the spot Ji SNP mark
Note can be used for the correlative study of the analyses such as spot Ji population genetic diversity level, Genetic Constitution of Population.
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