CN105238859B - A kind of method for obtaining chicken full-length genome high density SNP marker site - Google Patents
A kind of method for obtaining chicken full-length genome high density SNP marker site Download PDFInfo
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Abstract
The invention belongs to gene engineering technology field, there is provided a kind of method for obtaining chicken full-length genome high density SNP marker site, comprise the following steps：(1) the endonuclease bamhi distribution situation obtained with EcoRI and MseI double digestion chicken genome is predicted；(2) universal joint, bar code joint and pcr amplification primer thing are designed according to EcoRI and MseI endonuclease bamhi characteristic distributions；(3) build and simplify gene order-checking library；(4) library built using step (3) carries out upper machine sequencing；(5) SNP marker site is obtained according to sequencing result.A kind of general strategy is provided using double digestion GBS structure full-length genome high density SNP collection of illustrative plates so that obtain the cost in each SNP marker site reduces an order of magnitude than traditional die technology, and this method is consistent, and repeatability is high for the chicken of different cultivars.
Description
Technical field
The present invention relates to biological technical field, specifically, it is complete to be related to a kind of acquisition chicken based on sequencing genotyping technique
The method in genome high-density SNP marker site.
Background technology
As the model organism of birds, chicken turned into first agricultural economical animal for completing full genome sequencing in 2004,
Because different chicken breeds has huge bio-diversity, it is applied more and more as high-quality Genetics Model
To the field such as Quantitative Genetics and molecular breeding, functional gene positioning, the regulation and control of gene and development.Molecular labeling is research biology
The important tool of hereditary variation, SNP (Single nucleotide polymorphisms, SNP) are used as the
Three generations's molecular labeling, have the characteristics that quantity is more, distribution is wide, inheritance stability, be widely used in linkage analysis, full-length genome closes
The genetic breeding field such as connection analysis and gene group selection.Therefore understand the SNP information in chicken genome, be the genetic breeding mistake of chicken
Unusual the key link in journey.
The chicken full-length genome SNP typing methods of main flow mainly have two methods of gene typing chips and two generations sequencing at present.
The characteristics of gene typing chips is consistent, and as a result repetitive rate is high, but the cost of one experiment sample of chip technology parting is very
Height, for population genetic study field, the cost price of colony's parting is too big, and chip technology is limited by technology, also
There is SNP polymorphic sites in different groups poor universality, mark density is low, and (the SNP chip density of chicken main flow is at present
60kSNP chips), it is impossible to the problems such as meeting fine functional gene positioning and whole-genome association.Sequencing technologies of future generation
Development enables the research of genomics and transcription group more to go deep into, and sequencing can obtain the horizontal highly dense scale of full-length genome
Remember collection of illustrative plates, but simultaneously there is also unit sample cost it is too high the shortcomings that.
Simplify genomic sequencing technique (reduced-representation sequencing) and cause population analysis research
The identification of the high flux molecular labeling of required covering full-length genome is possibly realized with parting.But different simplification gene order-checking
Method build storehouse strategy, combination selection, the selection etc. of microarray dataset of single endonuclease digestion/double digestion have bigger difference, these
The efficiency and cost of follow-up parting will be significantly affected.For example, the method that RAD is sequenced builds storehouse strategy complexity, excessively
Step can disturb subsequent experimental result；Different restriction enzymes digestion frequency and distribution in different species gene groups is equal
Have relatively big difference, for particular species, from which kind of enzyme test just turn into determine experiment obtain SNP quantity and cost certainly
Determine factor；2b-RAD technologies use II Type B restriction enzyme, but the clip size of this digestion only has 25-35bp, 2b-RAD
Although technology can obtain the horizontal endonuclease bamhi of full-length genome, the frequency to be made a variation according to full-length genome, too short digestion piece
Section is difficult to be rich in SNP sites, causes mass data to lose, simultaneously because endonuclease bamhi is too short, can also bring many in genome
The mistake that repeat region compares so that SNP parting reliabilities decline to a great extent, severe jamming downstream application.
It is therefore desirable to develop a kind of analysis method in new chicken genome SNP marker site, there is provided be adapted to chicken genome
The enzymes combinations of SNP marker Locus Analysis in Shoots, to reduce the cost of Genotyping, provided just for the downstream application after Genotyping
Profit.
The content of the invention
In view of the shortcomings of the prior art, chicken is obtained based on sequencing genotyping technique it is an object of the invention to provide one kind
The method in full-length genome high density SNP marker site.
Sequencing Genotyping (Genotyping By Sequencing, GBS) technology is the Elshire by Cornell University
Et al. exploitation, its banking process is the simplest, and DNA is after digestion, given joint in connection, by during controlling PCR
The time of extension, to select the part of 100-500bp in digestion products, so as to realize the purpose of simplified gene order-checking；But
The defects of this method, is that not only amplification efficiency is higher during Jian Ku for small fragment digestion products, in the template of sequenator
Also very fast growth in amplification, the problem of part is less, the quality of data is poor can be utilized by easily causing sequencing data.
The invention provides a kind of acquisition chicken full-length genome high density SNP marker site based on sequencing genotyping technique
Method, comprise the following steps：
(1) the endonuclease bamhi distribution situation obtained with EcoRI and MseI double digestion chicken genome is predicted；
(2) universal joint, bar code joint and PCR is designed according to EcoRI and MseI endonuclease bamhi characteristic distributions to expand
Primer；
(3) build and simplify gene order-checking library；
(4) library built using step (3) carries out upper machine sequencing；
(5) SNP marker site is obtained according to sequencing result.
Optionally, the universal joint described in step (2) carries and restriction enzyme MseI identical cohesive end sequences
Row, described bar code joint carry and restriction enzyme EcoRI identical cohesive end sequences.
Optionally, the universal joint is by SEQ ID NO：1 and SEQ ID NO：Sequence anneals shown in 2 are formed double
Chain DNA, wherein SEQ ID NO：1 passes through 5 ' phosphorylation modifications.
Optionally, the bar code joint is by SEQ ID NO：3 and SEQ ID NO：What sequence anneals shown in 4 were formed
Double-stranded DNA；Wherein SEQ ID NO：4 pass through 5 ' phosphorylation modifications, SEQ ID NO：3 and SEQ ID NO：N and m in 4 are represented
Length is 6-9bp any short nucleotides bar code sequence.
Optionally, such as SEQ ID NO of the pcr amplification primer thing described in step (2)：5 and SEQ ID NO：Shown in 6.
Optionally, comprise the following steps in step (3)：
(a) combine EcoRI-MseI using restriction enzyme and digestion is carried out to chicken genome；
(b) universal joint and bar code joint are prepared；
(c) universal joint and bar code joint are mixed to form joint mixture by a certain percentage, then by itself and enzyme
Cut product and be attached reaction, obtain connection product；
(d) connection product equal proportion is subjected to mixed pond, obtains the connection product behind mixed pond；
(e) magnetic bead progress the first purifying acquisition first that 1.2-1.4 times of volume is added in the connection product behind mixed pond is pure
Change product；
(f) magnetic bead progress the second purifying acquisition second that 0.8-0.9 times of volume is added in first purified product is pure
Change product；
(g) enter performing PCR amplification to the second purified product and obtain PCR primer；
(h) magnetic bead that 1.2-1.4 times of volume is added in PCR primer carries out the 3rd purifying the 3rd purified product of acquisition；
(i) magnetic bead that 0.8-0.9 times of volume is added in the 3rd purified product carries out the 4th simplified genome of purifying acquisition
Sequencing library.
Optionally, the step of first purifying is with the 3rd purifying is identical, specifically includes：After adding magnetic bead, in gyroscope
System after upper incubation at room temperature 18-22min is incubated；Incubation is placed on supernatant discarding on magnetic frame after terminating, add 480-520
μ L 70% ethanol, slowly rotated after standing 30-40s, magnetic bead is moved on tube wall, after solution clarification, remove supernatant
Liquid, repeat this step and once precipitated；Low TE are added in the precipitation obtained again, are inhaled up and down with pipettor after beating,
10s is vibrated, clarification is stood after centrifugation and obtains supernatant；Wherein, it is relative to precipitation, Low TE addition described in 100 μ L
140-160μL。
Optionally, the step of the second purifying is with the 4th purifying is identical, specifically includes：After adding magnetic bead, in gyroscope upper chamber
Temperature is incubated 13-16min；Incubation is placed on supernatant discarding on magnetic frame after terminating, add 480-520 μ L 70% ethanol, stands
Slowly rotated after 30-40s, magnetic bead is moved on tube wall, after solution clarification, remove supernatant, repeated this step and once obtain
It must precipitate；Low TE are added in the precipitation obtained again, is inhaled up and down with pipettor after beating, is vibrated 10s, clarification is stood after centrifugation
Obtain supernatant；Wherein, relative to precipitation described in 100 μ L, Low TE addition is 30-50 μ L.
Optionally, the annealing system of the universal joint described in step (c) is：100μM SEQ ID NO:15μL；100μM
SEQ ID NO:25 μ L, 5 × Annealing Buffer 10 μ L, the μ L of nuclease-free water 30；Cycle of annealing is：95 DEG C are heated to,
And 25 DEG C are cooled to 1 DEG C/min speed, 25 DEG C of insulation 30min are after 4 DEG C of preservations.
The annealing system of bar code joint is：100μM SEQ ID NO:35μL；100μM SEQ ID NO:45 μ L, 5 ×
The μ L of Annealing Buffer 10, the μ L of nuclease-free water 30；Response procedures are：95 DEG C of 3min, cooled with 1 DEG C/min speed,
Until dropping to 25 DEG C, 25 DEG C of insulation 30min are after 4 DEG C of preservations.
The system Adapters Mix of joint mixing：The μ L of universal joint 0.8, the μ L of bar code joint 15, nuclease-free water
84.2 μ L, the μ L of total system 100.
Optionally, the system of the coupled reaction described in step (c) is：Digestion products 20 μ L, 5 × DNA Ligase
The μ L of Reaction Buffer 8, the μ L of DNA ligase 2, the μ L of nuclease-free water 5, the μ L of joint mixture 5；PCR is placed in after mixing
On, response procedures are：22 DEG C of insulation 1h, 65 DEG C of insulation 30min, are cooled to 4 DEG C of preservations.
The present invention develops a kind of method for being sequenced based on EcoR I-Mse I double digestions and carrying out Genotyping, is different product
The chicken of kind provides a kind of general strategy using double digestion GBS structure full-length genome high density SNP collection of illustrative plates so that obtains every
The cost in individual SNP marker site reduces an order of magnitude than traditional die technology, and this method is consistent, and repeatability is high.
Brief description of the drawings
Fig. 1 is the testing results of sequencing library Agilent 2100 provided by the invention.
Fig. 2 reports for sequencing quality provided by the invention.
Embodiment
The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It is it will be appreciated that following real
Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this area
Art personnel can carry out various modifications and replacement in the case of without departing substantially from spirit of the invention and spirit to the present invention.
The sequencing kit used in following examples is NextSeq 500High Output Kit (75cycles).
The software that the sequencing data used in following examples compares genome is bowtie2 (version number bowtie2-
2.2.3) (it is based on (SuSE) Linux OS), SNP identifies that software is Tassel (version number tassel-4.3.13).
Embodiment 1
Embodiment 1 is used to illustrate method of the present invention
1st, experiment material：
Red Jungle-fowl, the white Leghorn of business layer breed are gathered, commercial meat chickens kind Chinese mugwort pulls out increasingly broiler chicken, Lingnan Yellow chicken,
Chinese Native Chicken Breeds Huiyang beard chicken, Wenchang Chicken, Henan cockfighting, Qingyuan Chicken, black langshan chicken, camellia chicken, Beijing Fatty Chicken, Tibetan
Chicken, Silky fowl, shouguang chicken, bamboo silk chicken, the miscellaneous chickens of Shi Qi, Xianju Chicken, stealthy Cold boiled chicken, each 4-6 individual blood of yellow dwarf chickens
Sample, 96 individuals altogether, genome is extracted, and it is standby that genome concentration is diluted into 50ng/ μ L.
2nd, joint and primer sequence：
Synthesize a pair of universal linker sequences, 96 pairs of bar code joint sequences, one couple of PCR primers sequence.
3rd, sequencing library is built：
96 chicken sample extraction postgenomes are subjected to genome digestion.Reaction system is 20 μ L, including 15 μ L
Nuclease-free water, 2 μ L10 × CutSmart Buffer, the μ L enzymes 2 of 0.5 μ L enzymes 1,0.5 (0.5 μ L EcoR I,
0.5 μ L Mse I), 200ng sample DNAs, mix, centrifugation, be placed in PCR instrument, reaction condition is：37 DEG C of 90min, 65 DEG C of 30min,
4 DEG C of preservations.
4th, joint annealing is with mixing：
Universal joint reaction system is：Totally 50 μ L, including 30 μ L nuclease-free waters, 10 5 × Annealing of μ L
Buffer, SEQ ID NO：1 (100 μM) 5 μ L, SEQ ID NO：2 (100 μM) 5 μ L, mix centrifugation, and reaction condition is 95 DEG C
3min, decline 1 DEG C/min, until dropping to 25 DEG C, 25 DEG C of 30min, 4 DEG C preservations.
Bar code joint reaction system is：Totally 50 μ L, including 30 μ L nuclease-free waters, 10 5 × Annealing of μ L
Buffer, SEQ ID NO：3 (100 μM) 5 μ L, SEQ ID NO：4 (100 μM) 5 μ L, mix centrifugation, and reaction condition is 95 DEG C
3min, decline 1 DEG C/min, until dropping to 25 DEG C, 25 DEG C of 30min, 4 DEG C preservations.
The system Adapters Mix of joint mixing：The μ L of universal joint 0.8, the μ L of bar code joint 15, nuclease-free water
84.2 μ L, the μ L of total system 100.
5th, joint connects：
Reaction system is 40 μ L, including 20 μ L digestion products, 5 μ LNuclease-free water, 8 μ L 5 ×
DNALigase Reaction Buffer, 2 μ LExpressLink T4 DNALigase, 5 μ L Adapters Mix, fully
Mix, centrifugation, reaction condition is 22 DEG C of insulations 1h, 65 DEG C of 30min, and 4 DEG C preserve.
6th, pond is mixed：
Each 5 μ L in the connection product of 96 samples is admixed together, take out 240 μ L and purified for lower step.
7th, magnetic beads for purifying connection product：
312 μ L AMPure XP Beads are added in 240 μ L connection products, centrifuge tube are placed on gyroscope, 15-25
DEG C be incubated 20min, be then placed into 3min on magnetic frame, abandon supernatant；500 μ L70% ethanol are added, centrifuge tube is placed in magnetic force
On frame, slowly rotary tube, rotation take two turns after 30s, magnetic bead is moved on tube wall, after solution clarification, remove supernatant, so
This step is repeated once afterwards；Centrifuge tube is removed, of short duration centrifugation, centrifuge tube is positioned on magnetic frame, is removed with small pipette tips residual
Ethanol is stayed, dries 3min；150 μ L Low TE are added, is inhaled and beaten several times up and down with pipette tips, shake 10s, of short duration centrifugation is placed in magnetic force
On frame, 3min, after solution clarification, supernatant is transferred in new centrifuge tube；Added into 150 μ L Low TE eluents
120 μ L AMPure XP Beads, centrifuge tube is placed on gyroscope, 15-25 DEG C of incubation 15min, is then placed into magnetic frame
Upper 3min, abandons supernatant；500 μ L70% ethanol are added, centrifuge tube is placed on magnetic frame, slowly rotary tube after 30s, rotation
Two circles, make magnetic bead be moved on tube wall, after solution clarification, remove supernatant, then repeat this step once；Remove centrifugation
Pipe, of short duration centrifugation, centrifuge tube is positioned on magnetic frame, is removed residual ethanol with small pipette tips, is dried 3min；Add 50 μ L Low
TE, inhaled and beaten several times up and down with pipette tips, shaken 10s, of short duration centrifugation, be placed on magnetic frame, 3min, after solution clarification, by supernatant
Liquid is transferred in new centrifuge tube, then centrifuge tube is placed in into 2min on magnetic frame, and supernatant is transferred to new centrifuge tube, is obtained pure
Connection product after change.
Concentration mensuration and PCR amplifications.Qubit 2.0 determines connection product concentration after purification, to determine that PCR processes are pure
The amount of connection product after change.Amplification system is 60 μ L, including 50 μ L Platinum PCR SuperMix High
The connection product of Fidelity, 10ng after purification, 1.2 μ L10 μM Primer A, 1.2 μ L10 μM Primer B, mend free nucleic acid
For enzyme water to 60 μ L, reaction condition is 95 DEG C of 5min, 17 × (95 DEG C of 30s, 62 DEG C of 30s, 68 DEG C of 30s), 72 DEG C of 5min, and 4 DEG C are protected
Deposit.
Repeat step 5) purifying, finally eluted with 30 μ L Low TE.Qubit 2.0 determines library concentration, Agilent
2100 detection library fragments size distributions.Examining report is shown in accompanying drawing 1.8th, the selection of microarray dataset：
Using the NextSeq500 sequencing systems of Illumina bis- generations microarray datasets, single-ended 75bp sequencing kits are used.
Because NextSeq500 sequenators single can produce 400M sequencing reads, therefore the test platform and method maximizing
Sequencing cost is reduced, relative to Hiseq sequencing systems speed also faster.Accompanying drawing 2 is shown in sequencing quality report.
9th, the mining analysis of SNP marker：
SNP excavation is carried out to sequencing data using TASSEL softwares, genome mapping softwares are entered using bowtie2
OK.291,772 SNP markers are detected altogether, and the analysis of position distribution and functional annotation are carried out to it after detecting SNP, it is known that
SNP is evenly distributed on genome, the results detailed in Table 1, illustrates that using analysis method provided by the present invention data can be obtained
The high SNP marker Locus Analysis in Shoots result of quality.
Distribution and annotation result of the table 1SNP sites in coloured differently body.
| Chromosome | Chromosome length | SNP numbers | SNP spacing (bp) |
| 1 | 195276750 | 58839 | 3318 |
| 2 | 148809762 | 45818 | 3247 |
| 3 | 110447801 | 34272 | 3222 |
| 4 | 90216835 | 38865 | 3125 |
| 5 | 59580361 | 19024 | 3131 |
| 6 | 34951654 | 11675 | 2993 |
| 7 | 36245040 | 11281 | 3212 |
| 8 | 28767244 | 8776 | 3277 |
| 9 | 23441680 | 7518 | 3118 |
| 10 | 19911089 | 6244 | 3188 |
| 11 | 19401079 | 5881 | 3298 |
| 12 | 19897011 | 5958 | 3339 |
| 13 | 17760035 | 5256 | 3379 |
| 14 | 15161805 | 4337 | 3495 |
| 15 | 12656803 | 3616 | 3500 |
| 16 | 535270 | 134 | 3994 |
| 17 | 10454150 | 2749 | 3802 |
| 18 | 11219875 | 3913 | 3851 |
| 19 | 9983394 | 2658 | 3755 |
| 20 | 14302601 | 3914 | 3654 |
| 21 | 6802778 | 1983 | 3430 |
| 22 | 4081097 | 825 | 4946 |
| 23 | 5723239 | 1568 | 3650 |
| 24 | 6323281 | 1533 | 4124 |
| 25 | 2191139 | 401 | 5464 |
| 26 | 5329985 | 1123 | 4746 |
| 27 | 5209285 | 1176 | 4429 |
| 28 | 4742627 | 1073 | 4419 |
| Z | 82363669 | 12313 | 6689 |
| W | 1248174 | 49 | 25472 |
| It is total | 1003035513 | 291772 | 3437 |
The traditional die method of comparative example 1 obtains and chicken genome SNP marker
Comparative example 1 is the reference examples of embodiment 1.Sample is same as Example 1 in comparative example 1, and all samples are all used
The 60KSNP chips of Illumina companies chicken carry out Genotyping.The hybridization of chip, Scanning Detction work by Canada
DNALandmarks companies (DNA Landmarks Inc., Quebec, Canada) complete.By Quality Control, last residue 47,
965 SNP can be used for next step data analysis.The fund cost that each SNP is obtained is about 10 times in embodiment 1, during experiment
Between cost be about 5 times in embodiment 1.
The selection of the most suitable restriction endonuclease combination of the chicken genome of embodiment 2
Embodiment 2 is used to illustrate enzymes combinations used in the present invention.
Inventor considers different restriction enzyme site recognition points (such as identifying base number, G/C content, methylation status), sets altogether
8 groups of double digestion combinations are counted, are tested by 3 Lingnan Yellow chickens from the sequencing of the different enzymes combinations of Huiyang beard chicken individuals progress, it is real
Flow is tested with embodiment 1, experimental result is as shown in table 2.Understand, SNP number of EcoR I-Mse I enzymes combinations is 134,291
Individual (SNP number can change with the change of experimental subjects number), endonuclease bamhi number are 414,294, the ratio with genome
To rate highest, the typing assay of 96 samples can be carried out in a Nextseq500 sequencing experiment, input-output ratio is tested and reaches
To optimal.
2 different enzymes combinations of table genotyping result in chicken genome
| Enzymes combinations | SNP number | Endonuclease bamhi | Comparison rate |
| Pst I–Mse I | 402,083 | 1,247,742 | 97.26% |
| Pst I–ApeK I | 195,960 | 761,797 | 96.90% |
| EcoR I–Mse I | 134,291 | 414,294 | 98.37% |
| Bgl II–ApeK I | 133,770 | 436,503 | 97.82% |
| Pst I–Mse I | 117,571 | 498,114 | 94.28% |
| HinP1 I-Mse I | 94,724 | 491,451 | 95.03% |
| HinP1 I-ApeK I | 71,751 | 389,479 | 91.77% |
| EcoR I–Mse I | 26,112 | 96,527 | 96.25% |
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (1)
- A kind of 1. method for obtaining chicken full-length genome high density SNP marker site, it is characterised in that comprise the following steps：(1) the endonuclease bamhi distribution situation that prediction is obtained with EcoRI and MseI double digestion chicken genomes；(2) universal joint, bar code joint and pcr amplification primer thing are designed according to EcoRI and MseI endonuclease bamhi characteristic distributions；(3) build and simplify gene order-checking library；(4) library built using step (3) carries out upper machine sequencing；(5) SNP marker site is obtained according to sequencing result；Universal joint described in step (2) carries and restriction enzyme MseI identical cohesive end sequences, described bar Shape code joint carries and restriction enzyme EcoRI identical cohesive end sequences；The universal joint is by SEQ ID NO：1 and SEQ ID NO：The double-stranded DNA that sequence anneals shown in 2 are formed, wherein SEQ ID NO：1 passes through 5 ' phosphorylation modifications；The bar code joint is by SEQ ID NO：3 and SEQ ID NO：The double-stranded DNA that sequence anneals shown in 4 are formed；Wherein SEQ ID NO：4 pass through 5 ' phosphorylation modifications, SEQ ID NO：3 and SEQ ID NO：N and m in 4 represent that length is 6-9bp's Any short nucleotides bar code sequence；Pcr amplification primer thing such as SEQ ID NO described in step (2)：5 and SEQ ID NO：Shown in 6；Step comprises the following steps in (3)：(a) combine EcoRI-MseI using restriction enzyme and digestion is carried out to chicken genome；(b) universal joint and bar code joint are prepared；(c) universal joint and bar code joint are mixed to form joint mixture by a certain percentage, then produced itself and digestion Thing is attached reaction, obtains connection product；(d) connection product equal proportion is subjected to mixed pond, obtains the connection product behind mixed pond；(e) magnetic bead that 1.2-1.4 times of volume is added in the connection product behind mixed pond carries out first purifying acquisition the first purifying production Thing；(f) magnetic bead that 0.8-0.9 times of volume is added in first purified product carries out second purifying acquisition the second purifying production Thing；(g) enter performing PCR amplification to the second purified product and obtain PCR primer；(h) magnetic bead that 1.2-1.4 times of volume is added in PCR primer carries out the 3rd purifying the 3rd purified product of acquisition；(i) magnetic bead that 0.8-0.9 times of volume is added in the 3rd purified product carries out the 4th simplified gene order-checking of purifying acquisition Library；The step of first purifying is with the 3rd purifying is identical, specifically includes：After adding magnetic bead, it is incubated at room temperature on gyroscope System after 18-22min is incubated；Incubation is placed on supernatant discarding on magnetic frame after terminating, add 480-520 μ L 70% second Alcohol, slowly rotated after standing 30-40s, magnetic bead is moved on tube wall, after solution clarification, removed supernatant, repeat this step Suddenly once precipitated；Low TE are added in the precipitation obtained again, is inhaled with pipettor and is vibrated after beating up and down, stood after centrifugation Clarification obtains supernatant；Wherein, relative to precipitation described in 100 μ L, Low TE addition is 140-160 μ L；The step of second purifying is with the 4th purifying is identical, specifically includes：After adding magnetic bead, 13- is incubated at room temperature on gyroscope 16min；Incubation is placed on supernatant discarding on magnetic frame after terminating, add 480-520 μ L 70% ethanol, delays after standing 30-40s Slow rotation, makes magnetic bead be moved on tube wall, after solution clarification, removes supernatant, repeats this step and is once precipitated；Exist again Low TE are added in the precipitation obtained, is inhaled with pipettor and is vibrated after beating up and down, clarification is stood after centrifugation and obtains supernatant；Its In, relative to precipitation described in 100 μ L, Low TE addition is 30-50 μ L；The annealing system of universal joint described in step (b) is：100μM SEQ ID NO:1 5μL；100μM SEQ ID NO:25 μ L, 5 × Annealing Buffer 10 μ L, the μ L of nuclease-free water 30；Cycle of annealing is：95 DEG C are heated to, and with 1 DEG C/min speed is cooled to 25 DEG C, 25 DEG C of insulation 30min are after 4 DEG C of preservations；The annealing system of bar code joint is：100μM SEQ ID NO:3 5μL；100μM SEQ ID NO:45 μ L, 5 × The μ L of Annealing Buffer 10, the μ L of nuclease-free water 30；Response procedures are：95 DEG C of 3min, cooled with 1 DEG C/min speed, Until dropping to 25 DEG C, 25 DEG C of insulation 30min are after 4 DEG C of preservations；The system Adapters Mix of step (c) center tap mixing：The μ L of universal joint 0.8, the μ L of bar code joint 15, nuclease free The μ L of water 84.2, the μ L of total system 100；The system of coupled reaction described in step (c) is：μ L, the 5 × DNA Ligase Reaction of digestion products 20 The μ L of Buffer 8, the μ L of DNA ligase 2, the μ L of nuclease-free water 5, the μ L of joint mixture 5；It is placed in after mixing on PCR, response procedures For：22 DEG C of insulation 1h, 65 DEG C of insulation 30min, are cooled to 4 DEG C of preservations.
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| CN105671174B (en) * | 2016-03-14 | 2019-04-09 | 中国农业大学 | Chicken crawls character gene and to crawl the relevant DNA molecular marker of character to chicken |
| CN106191253B (en) * | 2016-07-14 | 2019-04-16 | 中国农业大学 | Beijing duck based on GBS technology simplifies gene order surveying method |
| CN106434867A (en) * | 2016-07-28 | 2017-02-22 | 江苏省家禽科学研究所 | SNP mark screening method for monitoring breed conservation effect of poultry, application thereof to chicken breed conservation and authentication method of SNP mark |
| CN111225986B (en) * | 2017-10-10 | 2021-02-05 | 中国农业科学院北京畜牧兽医研究所 | Chicken whole genome SNP chip and application thereof |
| CN110452990B (en) * | 2018-05-07 | 2021-03-23 | 华中农业大学 | SNP molecular marker for selecting laying rate of hens in later laying period and application thereof |
| CN108913797A (en) * | 2018-06-22 | 2018-11-30 | 中国农业科学院蔬菜花卉研究所 | The method that GBS obtains Chinese cabbage group genome SNP building finger-print |
| CN113039288B (en) * | 2018-09-29 | 2022-04-26 | 中国农业大学 | Whole-genome SNP chip for laying hens and application thereof |
| CN109680041A (en) * | 2018-12-25 | 2019-04-26 | 上海派森诺生物科技股份有限公司 | A kind of processing method based on the sequencing sample for simplifying gene order-checking |
| CN113322333B (en) * | 2021-07-06 | 2022-07-05 | 广西大学 | CNV molecular marker combination related to Guangxi hemp chicken body size and slaughter traits based on whole genome sequencing screening and application |
| CN114480673B (en) * | 2022-03-04 | 2022-12-23 | 江苏省家禽科学研究所 | Chicken low-density SNP liquid phase chip based on targeted capture sequencing and application thereof |
| CN115679012B (en) * | 2022-10-18 | 2023-07-04 | 武汉市农业科学院 | Chilli whole genome SNP-Panel and application thereof |
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