CN108913797A - The method that GBS obtains Chinese cabbage group genome SNP building finger-print - Google Patents
The method that GBS obtains Chinese cabbage group genome SNP building finger-print Download PDFInfo
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Abstract
The present invention provides a kind of method for obtaining Chinese cabbage group full-length genome high density SNP site building finger-print based on GBS technology, including：(1) different digestion schemes is designed, predicts single endonuclease digestion or double digestion cabbage material genome endonuclease bamhi distribution situation obtained；(2) bar code connector and PCR amplification primer are designed according to endonuclease bamhi characteristic distributions；(3) it constructs and simplifies gene order-checking library；(4) upper machine sequencing is carried out using the library that step (3) construct；(5) SNP marker site is obtained according to sequencing result, for constructing finger-print.The present invention constructs full-length genome high density SNP map for different Chinese cabbage group material use GBS and provides a kind of general policies, this method reduce sequencing costs, restriction enzyme site is evenly distributed in the genome, 100 parts of cabbage materials are sequenced using this method, expense can reduce by 70% or so, and the polymorphic site of acquisition is sufficiently used for character analysis.
Description
Technical field
The present invention relates to gene sequencing technologies, specifically, being related to a kind of based on the GBS technology acquisition full base of Chinese cabbage group
Because of a group method for high density SNP site building finger-print.
Background technique
The full-length genome SNP typing method of mainstream mainly has two methods of gene typing chips and two generations sequencing at present.Base
Because the characteristics of typing chip be it is consistent, as a result repetitive rate is high, but the cost of one experiment sample of chip technology parting is very high,
For population genetic study field, the cost price of group's parting is too big, and chip technology is limited by technology, there is also
SNP polymorphic site poor universality in different groups, mark density is low, is not able to satisfy the positioning of fine functional gene and full genome
The problems such as group association analysis.
Whole genome sequencing (WGS), i.e. genome sequencing, are sequenced crop complete genome DNA
Analysis reaches expected sequencing depth, and sequencing amount is larger, costly.Genotyping technique Genotyping by is sequenced
Sequencing (GBS), be it is a kind of interrupt genomic DNA fragment using restriction enzyme, endonuclease bamhi both ends are carried out high
Flux sequencing sufficiently represents the sequencing of target species full-length genome information to obtain a large amount of genetic polymorphism sequence labels
Strategy, this method experimental procedure is simple, at low cost, and can not depend on and can obtain within the scope of full-length genome with reference to genome
Genetic polymorphism label.But since different plant species genome is different, most suitable restriction enzyme processing mode is also different
Sample.Different restriction enzymes digestion frequency and distribution in different species gene groups have relatively big difference, for specific
Species select which kind of restriction endonuclease test just becoming and determines that experiment obtains SNP quantity, and SNP is evenly distributed degree in genome
And spend the determinant of cost.
Summary of the invention
The object of the present invention is to provide one kind to obtain Chinese cabbage group full-length genome high density SNP site based on GBS technology
The method for constructing finger-print.
In order to achieve the object of the present invention, the present invention use 10 kinds of digestion schemes, handle two kinds of cabbage materials (Chinese cabbage and
Pakchoi), every part of material takes 5 single plants, totally 10 samples, carries out GBS analysis, explores and is most suitable for Chinese cabbage group GBS analysis
Digestion scheme.
The present invention is based on the sides that GBS technology obtains Chinese cabbage group full-length genome high density SNP site building finger-print
Method includes the following steps：
(1) according to the digestion scheme of table 1, single endonuclease digestion or double digestion cabbage material genome digestion piece obtained are predicted
Section distribution situation；
(2) bar code connector and PCR amplification primer are designed according to endonuclease bamhi characteristic distributions；
(3) it constructs and simplifies gene order-checking library；
(4) upper machine sequencing is carried out using the library that step (3) construct；
(5) SNP marker site is obtained according to sequencing result, for constructing finger-print.
1 digestion scheme of table
Preferably, the base sequence of step (2) the bar code connector is：
Normal chain：5′-AATGATACGGCGACCACCGAGATCTACACXXXXXXXXACACTCTT
TCCCTACACGACGCTCTTCCGATCT-3′
Minus strand：5′-P-GATCGGAAGAGCACACGTCTGAACTCCAGTCACYYYYYYYYAT
CTCGTATGCCGTCTTCTGCTTG-3′
Wherein, the XXXXXXXX in the normal chain represents normal chain bar code sequence, and the YYYYYYYY in the minus strand is represented
Minus strand bar code sequence.
The normal chain bar code sequence XXXXXXXX and minus strand bar code sequence YYYYYYYY is selected from following any bar
Sequence：
Preferably, the base sequence of step (2) the PCR amplification primer is (SEQ ID NO:1-2)：
PF：5′-AATGATACGGCGACCACCGA-3′
PR：5′-CAAGCAGAAGACGGCATACG-3′
Method above-mentioned, step (3) include following sub-step：
(a) according to the digestion scheme of table 1, single endonuclease digestion or double digestion are carried out to cabbage material genome；
(b) endonuclease bamhi carries out 3 ' ends plus A processing, connects bar code connector；
(c) bar code connector is attached with digestion products and is reacted, obtain connection product；
(d) connection product equal proportion is subjected to mixed pond, the connection product after obtaining mixed pond；
(e) purifying concentration is carried out to the connection product behind mixed pond, obtains purified product；
(f) PCR amplification is carried out to purified product and obtains PCR product；
(g) PCR product carries out agarose gel electrophoresis, recycles target fragment to get gene order-checking library is simplified.
The present invention utilizes GBS technology, screens restriction enzyme site in Chinese cabbage by digestion according to the specificity of Chinese cabbage group
Be evenly distributed in class crop gene group, can be applied to identification Chinese cabbage group SNP site carry out fingerprint map analyzing, wherein A,
E, F, G or J are preferred digestion scheme.Best digestion scheme is E, i.e. RsaI+HaeIII enzymes combinations, and the digestion scheme is big
In Chinese cabbage and pakchoi sample, compared with other 9 kinds of digestion schemes, it can obtain more SNP sites (genotype), and digestion
Site is evenly distributed on Chinese cabbage group genome, provides authentic data support for follow-up test analysis.
Cabbage material used in the present invention is that section rich 80 and Hunan show short-foot are white.Section rich 80：Chinese cabbage, by ' it is green that head is embraced in Beijing '
It is selfed obtained by 10 generations, growth period 80-85 days, dark green upright, high stake is folded to embrace.With it for there are two parent's improved variety：It is green elegant and
(' it is green that head is embraced in Beijing ' is Chinese cabbage to green treasured, is seen《China's Vegetable kind will》, Vegetable & Flower Inst., Chinese Academy of Agriculture Science master
It compiles, at Chinese agriculture Science Press, June the 1st edition in 2001).Hunan show short-foot is white：Pakchoi (white stalk dish), by ' Nanjing short-foot
It is yellow ' selfing 10 generations gained, growth period is 45-50 days, and (' Nanjing short-foot is yellow ' is pakchoi to short-foot Huang type, is seen《China's Vegetable product
Kind will》, Vegetable & Flower Inst., Chinese Academy of Agriculture Science edits, at Chinese agriculture Science Press, June the 1st edition in 2001).
By above-mentioned technical proposal, the present invention at least has following advantages and beneficial effect：
The building of Chinese cabbage group full-length genome high density SNP site is obtained based on GBS technology the present invention provides one kind to refer to
The method of line map is that different Chinese cabbage group material use single endonuclease digestions or double digestion GBS construct full-length genome high density SNP figure
Spectrum provides a kind of general strategy, so that obtaining the cost in each SNP marker site reduces a number than traditional die technology
Magnitude, this method is consistent, and repeatability is high.
The present invention is directed to Chinese cabbage group, determines efficient digestion scheme, and screening is applied to building Chinese cabbage group fingerprint
The SNP site of map reduces sequencing cost, obtains sufficient amount of polymorphic site, is used for character analysis.Utilize this method pair
100 parts of cabbage materials are sequenced, and every part of material surveys 400Mb, every part 300 yuan of material, expense be 100 × 300 yuan/part=
30,000 yuan, and resurvey sequence every part of material sequencing depth 10 ×, every part of Master Cost 1,000 yuan, i.e., 100 × 1,000 yuan/part=
100,000 yuan, it can be seen that GBS specific gravity sequencing expense can reduce by 70% or so.Using RsaI+HaeIII enzymes combinations scheme into
Row GBS sequencing, every part of material obtain 80,000SNP or so, and SNP site is evenly distributed on cabbage genome, acquisition it is more
State property site is sufficiently used for character analysis.
Detailed description of the invention
Fig. 1 is to obtain genotype quantity based on 10 kinds of digestion schemes of GBS technology in present pre-ferred embodiments.
Fig. 2 is to obtain SNPs quantity based on 10 kinds of digestion schemes of GBS technology in present pre-ferred embodiments.
Fig. 3 A- Fig. 3 E is respectively digestion option A in present pre-ferred embodiments, E, F, G and J processing obtained gene of S1-1
The distribution situation of type on chromosome.
Fig. 4 is that sample room variant sites analyze result in present pre-ferred embodiments.
Fig. 5 is that similarity-rough set analysis is tied between different materials or same material difference sample in present pre-ferred embodiments
Fruit.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
According to conventional laboratory conditions, such as Sambrook molecular cloning experiment handbook (Sambrook J&Russell DW,
Molecular Cloning:A Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.
Embodiment 1 obtains the side of Chinese cabbage group full-length genome high density SNP site building finger-print based on GBS technology
Method
1, test material
Using 2 cabbage height generation selfing based materials in the present embodiment, i.e., (rich 80) of section are sample 1 (S1) and small to Chinese cabbage
Chinese cabbage (Hunan show short-foot is white) is sample 2 (S2), and each sample selects 5 single plants, i.e. S1-1, S1-2, S1-3, S1-4, S1-5,
S2-1,S2-2,S2-3,S2-4,S2-5。
2, digestion scheme
By simulating digestion, we select the uniform three kinds of restriction endonucleases of the position distribution of endonuclease bamhi on chromosome,
HaeIII,RsaI,and EcoRV-Carry out single endonuclease digestion or double digestion, and according to selecting different segments to carry out after digestion
Sequencing analysis, totally 10 kinds of digestion schemes, as shown in table 2.
2 10 kinds of digestion scheme informations of table
Note：Number of tags refers to specific inscribe enzymatic treatment Chinese cabbage postgenome, the number of fragments of specific fragment length.Enzyme
Cut efficiency：The amount of test of many times measurement restriction endonuclease certain time digested DNA fragmentation.
3, library construction and Illumina sequencing
To each sample gene group DNA, digestion processing is carried out according to different digestion schemes, reaction system is：50ng/μL
2 μ L of DNA, restriction endonuclease each 5U, 10 × reaction buffer 2 μ L, 20 μ L of total volume.Response procedures：37 DEG C 6 hours, 65 DEG C
90 minutes.Endonuclease bamhi carries out 3 ' ends plus A processing, is then attached reaction, the connector with different index sequences is distinguished
It is connected on each sample endonuclease bamhi.Reaction system is：25 μ L, 25pmol bar code connector of digestion products, the T4 of 50000U
Ligase (NEB).Reaction condition：16 DEG C overnight.Restriction enzyme site in connection product will be destroyed, to keep away after connecting connector
Exempt from digestion again.Generated mixed sequence is connected between non-complete degestion or two endonuclease bamhis in order to remove, and utilizes 1U's
Restriction endonuclease carries out secondary digestion.Every 12 products with different index are mixed into a sequencing library, with Qiagen reagent
After box carries out purifying concentration, it is separated by electrophoresis on 2% Ago-Gel.Each mixing sequencing library carries out in 50 μ L systems
PCR amplification, amplification system are：50-100ng digestion connection product, 1 × HF buffer；3.5mM MgCl2；0.4mM dNTPs；
1U amplification enzyme；5pmol amplimer (PF：AATGATACGGCGACCACCGA；PR：CAAGCAGA AGACGGCATACG), it is overall
30 μ L of product.
By PCR amplification, it can introduce at 5 ' ends and mutually be recruited with oligonucleotides fixed on Illumina flow cell
Pair sequence.PCR product is returned using agarose gel electrophoresis removal connector and primer sequence using Qiagen plastic recovery kit
Target fragment and constant volume are received to 25 μ L volumes.The sequencing library built Qubit (Invitrogen), Agilent 2100
(Agilent technology) and Real-time quantitative PCR (Bio-Rad) are quantitative, finally exist
It is sequenced on Illumina GA2 × and HiSeq4000 platform.Obtain high quality sequence, according to the index sequence at both ends and
Restriction enzyme site sequence is assigned in different samples, is used for subsequent analysis.
Above-mentioned 10 kinds of digestion schemes are selected, qualified each sample genomic DNA carries out digestion respectively to detection.To what is obtained
3 ' end of endonuclease bamhi progress plus A processing, PCR amplification, purifying, sample mixing, cut glue selection purpose at connection Dual-index sequence measuring joints
Segment is sequenced after library quality inspection is qualified with Illumina HiSeq4000.For the accuracy of assessment digestion experiment, day is selected
This fine rice is sequenced as control (Control), the control as sequencing data quality evaluation.
4, sequencing strategy and data quality accessment
It completes 10 samples and builds library sequencing, (both ends are respectively surveyed using Hiseq4000 sequencing instrument output PE150 data
150bp), starting sample carries out upper machine sequencing, every part of material selection endonuclease bamhi recovery scheme total 10 on same lane
Seed type.
When sequencing system is sequenced, chip preparation can be carried out to library first, it is therefore an objective to by library DNA (RNA) mould
Plate is fixed on chip, and during fixed dna template, each DNA (RNA) molecule will form a cluster, and a cluster is exactly
One sequencing site, physical location can overlap between minimal amount of cluster and cluster during being fixed, and in sequencing, survey
Sequence software is analyzed and is identified by the point of these overlappings of preceding 4 base-pairs, these are overlapped point position and is separated, is guaranteed each
What point measured is a DNA molecular, therefore sequencing sequence 5 ' holds the error rate of preceding several bases relatively high.In addition mistake is sequenced
Rate can be increased with the increase of sequencing sequence (Sequenced Reads) length, this is by chemical reagent in sequencing procedure
Caused by consumption.Therefore when carrying out the distributional analysis of base sequencing quality, the base Mass Distribution of sample preceding 4 bases with
The mass value of more than ten of base can be lower than intermediate sequencing base afterwards, but its mass value is all higher than Q30%.Using Q20 (%), Q30
(%) (calculating separately the percentage that base of the Phred numerical value greater than 20,30 accounts for overall base) and GC (%) (calculate bases G and C
Quantity summation account for the percentage of total base quantity) quality of data of sequencing output is assessed, the results are shown in Table 3.
3 data assessment of table statistics
Note：Digestion scheme：A~J；Sample number into spectrum：Two samples S1 and S2, each sample take 5 single plants, i.e. S1-1, S1-
2, S1-3, S1-4, S1-5, S2-1, S2-2, S2-3, S2-4, S2-5, OryzasativaLcv.Nipponbare rice is as control；Q20 (%)：Mass value is big
In the base percentage for being equal to 20；Q30 (%)：Mass value is more than or equal to 30 base percentage；GC (%)：Sample
G/C content.
5, raw sequencing data is analyzed
Firstly, low quality reads filtering is carried out to initial data using NGSQCToolkit_v2.3.3 software, secondly, being
Reliable variation information is obtained, reads is compared after filtering using BWA (Burrows-Wheeler Alignment) software
To Chinese cabbage with reference on genome, variation information extraction is carried out using Samtools software, and then extract VCF text using perl script
Part, it is final to obtain SNP message file (each variant sites at least 3 reads are supported).
6, interpretation of result
(1) Genotyping-by-sequencing reliability determination
In order to verify Genotyping-by-sequencing reliability, we are by Chinese cabbage material (5 lists in experiment
Strain) it has carried out resurveying sequence, i.e. reS1-1, reS1-2, reS1-3, reS1-4, reS1-5.We select in GBS in digestion option A
S1-1, S1-2, S1-3, the data of S1-4, S1-5 the results are shown in Table 4 compared with weight sequencing data carries out analysis.In 5 samples,
Two methods of the shared genotype locus of re-sequencing and GBS is 304,603, accounts for the consistent genotype (304,685) of the two
99.9% or more.For digestion option b~J, same conclusions are also obtained.Therefore, the data obtained by GBS are very reliable.
Table 4 analyzes GBS reliability between sequence reS1-1 and GBS S1-1 sample by resurveying
(2) quantity of SNPs and genotype between sample in GBS method
Every kind of digestion scheme survey 10 × data volume, but since different endonuclease digestion segment total numbers are different, finally
The quantity variance of each sample is very big.To exclude difference bring analytical error between data volume size, in analysis in each sample
150M data are randomly selected to be analyzed.By Fig. 1 and Fig. 2 it is found that being analyzed by genotype genotype and SNPs, option b and C
The genotype quantity of acquisition is considerably less than other digestion schemes.It is interesting that the genotype quantity that Chinese cabbage S1 material obtains is all
Higher than pakchoi S2 material.Distinguish uniformity coefficient and SNP quantitative analysis situation according to SNP site, digestion option A, E, F, G and
J is preferred embodiment, and the genotype quantity of acquisition is higher, other opposite digestion schemes have some superiority, especially digestion scheme E,
Highest polymorphism mark site is obtained, and the SNP site that the digestion scheme generates is evenly distributed on cabbage genome.
It is worth noting that digestion scheme G and J select all segments to carry out sequencing analysis；Digestion option A only selects a kind of restriction endonuclease,
And digestion scheme E and F respectively select two kinds of restriction endonucleases to be handled.
(3) between analysis different materials or with the similarity between a material difference sample
The S1-1 sample of digestion scheme E processing is randomly selected, the SNPs of the sample is evenly distributed on 10 chromosomes of Chinese cabbage
On, as shown in figure 3, the close expected digestion effect of selected restriction endonuclease.It is wherein distributed on A09 chromosome at most, is 2,850 SNPs
Site；SNPs quantity on A04 and A07 is only the half or so on A09.Digestion option A, E, F, G and J processing S1-1 are obtained
The distribution situation of genotype on chromosome is shown in Fig. 3 A- Fig. 3 E respectively.
In order to analyze the affiliation between different samples, We conducted similarity analysis between sample.
Similarity=(Section II kind SNPs quantity)/(I kind SNPs quantity) * 100%
As a result as shown in figure 4, during using analysis of biological information, the variation type of Section III and IV type is deleted, and is only protected
Stay I, II and III seed type.Similarity is the ratio of Section II kind SNPs quantity and I kind SNPs quantity.Pass through similitude ratio
Compared with discovery, similarity is below 40% between different materials, and the similarity between same material difference sample is higher than 90%, therefore GBS
Polymorphic site information obtained by method, can efficiently differentiate the Chinese cabbage material (Fig. 5) of separate sources, other digestion schemes also obtain
To similar result.
The effect of different digestion schemes is shown in Table 5 and (randomly selects, combine between same material difference sample：S1-1/S1-2,S1-
1/S1-4,S2-2/S2-4,S2-3/S2-5；It is combined between different materials：S1-2/S2-1,S1-4/S2-3,S1-3/S2-2,S1-5/
S2-5)。
The effect of the different digestion schemes of table 5
Distribution situation of the SNP site obtained by partially digested scheme (A, E, F, G and J) in different chromosomes is shown in Table 6- respectively
1 to table 6-5.
Distribution situation of the table 6-1 SNP site in different chromosomes
| Chromosome | SNP number (a) on every chromosome | SNP spacing (bp) |
| 1 | 2117 | 12754 |
| 2 | 1892 | 14376 |
| 3 | 2588 | 12172 |
| 4 | 1413 | 13800 |
| 5 | 2119 | 12034 |
| 6 | 2573 | 9716 |
| 7 | 2042 | 12733 |
| 8 | 1784 | 12052 |
| 9 | 2857 | 13651 |
| 10 | 1379 | 11965 |
| It is total | 20764 | 12459 |
Note：Data are derived from aS2-1 (by A digestion scheme, second sample of processing, the 1st single plant).To control data difference
The different influence to result, we only randomly select 150Mb data in each sample and analyze.
Distribution situation of the table 6-2 SNP site in different chromosomes
| Chromosome | SNP number (a) on every chromosome | SNP spacing (bp) |
| 1 | 2831 | 9537 |
| 2 | 2555 | 10646 |
| 3 | 3200 | 9844 |
| 4 | 1923 | 10140 |
| 5 | 2646 | 9637 |
| 6 | 2956 | 8457 |
| 7 | 2669 | 9741 |
| 8 | 2116 | 10161 |
| 9 | 3836 | 10167 |
| 10 | 1610 | 10248 |
| It is total | 26342 | 9821 |
Note：Data are derived from eS2-1 (by E digestion scheme, second sample of processing, the 1st single plant).To control data difference
The different influence to result, we only randomly select 150Mb data in each sample and analyze.
Distribution situation of the table 6-3 SNP site in different chromosomes
| Chromosome | SNP number (a) on every chromosome | SNP spacing (bp) |
| 1 | 2117 | 12754 |
| 2 | 1928 | 14108 |
| 3 | 2521 | 12495 |
| 4 | 1378 | 14151 |
| 5 | 2126 | 11994 |
| 6 | 2579 | 9694 |
| 7 | 2051 | 12677 |
| 8 | 1772 | 12133 |
| 9 | 2859 | 13641 |
| 10 | 1544 | 10687 |
| It is total | 20875 | 12393 |
Note：Data are derived from fS2-1 (by F digestion scheme, second sample of processing, the 1st single plant).To control data difference
The different influence to result, we only randomly select 150Mb data in each sample and analyze.
Distribution situation of the table 6-4 SNP site in different chromosomes
| Chromosome | SNP number (a) on every chromosome | SNP spacing (bp) |
| 1 | 1882 | 14346 |
| 2 | 1742 | 15614 |
| 3 | 2238 | 14075 |
| 4 | 1310 | 14885 |
| 5 | 1931 | 13206 |
| 6 | 2359 | 10598 |
| 7 | 1920 | 13542 |
| 8 | 1539 | 13970 |
| 9 | 2545 | 15324 |
| 10 | 1233 | 13382 |
| It is total | 18699 | 13835 |
Note：Data are derived from gS2-1 (by G digestion scheme, second sample of processing, the 1st single plant).To control data difference
The different influence to result, we only randomly select 150Mb data in each sample and analyze.
Distribution situation of the table 6-5 SNP site in different chromosomes
Note：Data are derived from jS2-1 (by J digestion scheme, second sample of processing, the 1st single plant).To control data difference
The different influence to result, we only randomly select 150Mb data in each sample and analyze.
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 modified or is improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Vegetable & Flower Inst., Chinese Academy of Agriculture Science
<120>The method that GBS obtains Chinese cabbage group genome SNP building finger-print
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Claims (7)
1. based on the method that GBS technology obtains Chinese cabbage group full-length genome high density SNP site building finger-print, feature
It is, includes the following steps：
(1) according to the digestion scheme of table 1, single endonuclease digestion or double digestion cabbage material genome endonuclease bamhi obtained point are predicted
Cloth situation；
(2) bar code connector and PCR amplification primer are designed according to endonuclease bamhi characteristic distributions；
(3) it constructs and simplifies gene order-checking library；
(4) upper machine sequencing is carried out using the library that step (3) construct；
(5) SNP marker site is obtained according to sequencing result, for constructing finger-print；
1 digestion scheme of table
2. the method according to claim 1, wherein the base sequence of step (2) the bar code connector is：
Normal chain：5′-AATGATACGGCGACCACCGAGATCTACACXXXXXXXXACACTCTTTCCCTACACGACGCTCTTCC
GATCT-3′
Minus strand：5′-P-GATCGGAAGAGCACACGTCTGAACTCCAGTCACYYYYYYYYATCTCGTATGCCGTCTTCTGCT
TG-3′
Wherein, the XXXXXXXX in the normal chain represents normal chain bar code sequence, and the YYYYYYYY in the minus strand represents minus strand
Bar code sequence.
3. according to the method described in claim 2, it is characterized in that, the normal chain bar code sequence XXXXXXXX and the minus strand
Bar code sequence YYYYYYYY is selected from following any bar sequence：
4. the method according to claim 1, wherein the base sequence of step (2) the PCR amplification primer is：
PF：5′-AATGATACGGCGACCACCGA-3′
PR：5′-CAAGCAGAAGACGGCATACG-3′.
5. the method according to claim 1, wherein step (3) includes following sub-step：
(a) according to the digestion scheme of table 1, single endonuclease digestion or double digestion are carried out to cabbage material genome；
(b) endonuclease bamhi carries out 3 ' ends plus A processing, connects bar code connector；
(c) bar code connector is attached with digestion products and is reacted, obtain connection product；
(d) connection product equal proportion is subjected to mixed pond, the connection product after obtaining mixed pond；
(e) purifying concentration is carried out to the connection product behind mixed pond, obtains purified product；
(f) PCR amplification is carried out to purified product and obtains PCR product；
(g) PCR product carries out agarose gel electrophoresis, recycles target fragment to get gene order-checking library is simplified.
6. the method according to claim 1, wherein using GBS technology, according to the specificity of Chinese cabbage group,
By digestion, screens restriction enzyme site and be evenly distributed on Chinese cabbage group genome, can be applied to the SNP of identification Chinese cabbage group
Site carries out fingerprint map analyzing, and preferred digestion scheme is A, E, F, G or J.
7. according to the method described in claim 6, it is characterized in that, the digestion scheme is E.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111883212A (en) * | 2020-02-19 | 2020-11-03 | 中国热带农业科学院热带生物技术研究所 | Construction method and construction device of DNA fingerprint spectrum and terminal equipment |
| CN114507728A (en) * | 2022-03-03 | 2022-05-17 | 苏州贝康医疗器械有限公司 | Capture primer and application thereof |
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| CN104694635A (en) * | 2015-02-12 | 2015-06-10 | 北京百迈客生物科技有限公司 | Method for constructing high-flux simplified genome sequencing library |
| CN105238859A (en) * | 2015-10-13 | 2016-01-13 | 中国农业大学 | Method for acquiring chicken whole genome high-density SNP marker sites |
| CN106191253A (en) * | 2016-07-14 | 2016-12-07 | 中国农业大学 | Beijing duck based on GBS technology simplifies gene order surveying method |
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| CN104694635A (en) * | 2015-02-12 | 2015-06-10 | 北京百迈客生物科技有限公司 | Method for constructing high-flux simplified genome sequencing library |
| CN105238859A (en) * | 2015-10-13 | 2016-01-13 | 中国农业大学 | Method for acquiring chicken whole genome high-density SNP marker sites |
| CN106191253A (en) * | 2016-07-14 | 2016-12-07 | 中国农业大学 | Beijing duck based on GBS technology simplifies gene order surveying method |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111883212A (en) * | 2020-02-19 | 2020-11-03 | 中国热带农业科学院热带生物技术研究所 | Construction method and construction device of DNA fingerprint spectrum and terminal equipment |
| CN111883212B (en) * | 2020-02-19 | 2021-11-26 | 中国热带农业科学院热带生物技术研究所 | Construction method and construction device of DNA fingerprint spectrum and terminal equipment |
| CN114507728A (en) * | 2022-03-03 | 2022-05-17 | 苏州贝康医疗器械有限公司 | Capture primer and application thereof |
| CN114507728B (en) * | 2022-03-03 | 2024-03-22 | 苏州贝康医疗器械有限公司 | Capturing primer and application thereof |
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Application publication date: 20181130 |