CN107686862A - A kind of method of freshwater fish screening microsatellite sequence - Google Patents
A kind of method of freshwater fish screening microsatellite sequence Download PDFInfo
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Abstract
Present invention relates particularly to separation and the screening technique that microsatellite sequence is carried out in freshwater fish.Belong to DNA molecular marker technology.The present invention uses Enrichment by Magnetic Beads microsatellite sequence, can simply, quickly filter out the microsatellite sequence in most freshwater fish whole gene groups, develop substantial amounts of micro-satellite primers in a short time.Freshwater fish microsatellite DNA technical system is established, makes these molecular labelings be applied to identify in fish germ plasma resource, the research of genetic breeding.
Description
Technical field
The present invention relates to DNA molecular marker technology, and in particular in freshwater fish carry out microsatellite sequence separation and
Screening technique.
Background technology
Microsatellite is that conventional variable number tandem repeat marks one of (VNTR), also known as simple tandem sequence repeats sequence
Row（Simple Sequence Repeat, SSR）Or simple sequence length is polymorphic（Simple Sequence Length
Polymorphism, SSLP）Or Short tandem repeatSTR（Short Tandem Repeats, STR）, it is one in DNA molecular
Fragment, with the motif of 1-6 base composition, such as（CA）n/（GT）n、（CAC）n/（GTG）N etc., into joining end to end tandem sequence repeats
It is distributed in whole gene group, typically long tens to hundreds of bp, its variable repeat number constitutes the basis of microsatellite polymorphism.
The length of usual polymorphic bandses is in 100-300bp.Each specific microsatellite locus forms by two parts：Middle core
Area and the flanking region of both sides.Core space contains the multicopy sequence of short series connection, and recurring unit's number changes at random, and flank
Sequence is relatively conservative in evolution.
Microsatellite technology is built upon the conventional DNA polymorphism analysis method on the basis of PCR, and microsatellite DNA repeats in itself
The variation of units is to form the basis of microsatellite locus polymorphism, precisely designed specific primer, the bar of PCR amplifications
Band non-overlapping copies, microsatellite amplified production are separated using polyacrylamide gel electrophoresis, and silver staining method dyeing, band is clear, more
State property information is accurate；Microsatellite technology repeatability is strong, and the sequence gradient on same film can provide resolution ratio up to 1 bp's
Fine difference, make microsatellite result accuracy high, be easy to be compared, fully show the superiority of microsatellite technology.Therefore it is micro-
Satellite technology also has important application prospect in the research of fish germ plasma resource.
Application of 1 microsatellite marker in terms of character analysis
Most of economic characters category quantitative characters, positioning analysis (QTL, Quantitative of the quantitative character on linkage map
Traits Loci) be agro-ecology genetic research importance, be molecular breeding subject basic research.QTL is positioned
Genetic analysis is carried out to character using collection of illustrative plates and mark, it is in hereditary evolution that the linkage analysis of character is prepared with genetic map
2 aspects of inseparable same problem.
Application of 2 microsatellite markers in terms of fish germplasm identification
Aquatic biological Germplasm Identification includes：The identification of different cultivars or different proles in identification, species between species, hybrid
Identification, paternity test etc..Due to fish microsatellite category codominant inheritance, the frequency of all allele can be accurately calculated,
Therefore there is certain superiority in above-mentioned Germplasm Identification.
Application of 3 microsatellite markers in terms of genetic breeding
Microsatellite marker be used for aquatic livestock breeding research mainly 2 aspect, when with microsatellite analyze colony, family and
The hereditary difference of individual, calculated using these differences between colony, the genetic distance between family between individual, it is true in conjunction with phenotype
This combo of becoming engaged scheme, reaches and both preferably goes out phenotype, avoids the purpose of inbred again；Second, according to character analysis knots such as QTL
Fruit, the excellent individual of character is filtered out using the marks such as the microsatellite related to character or gene, then established from excellent individual
Kind or strain.
The premise studied using micro-satellite labeling technique is to first have to obtain microsatellite sequence.Now more popular is micro-
Satellite DNA access approaches mainly obtain the positive colony containing microsatellite from genomic library.At present in plant and the food in one's mouth
It is more that the report of microsatellite sequence is screened in newborn animal, but due to aquatic biological and plant and the group of mammalian genomic sequences
Into and all difference the characteristics of microsatellite repetitive sequence, therefore when being applicable with Enrichment by Magnetic Beads microsatellite sequence
Need to adjust accordingly in the selection of probe when DNA extraction method and enrichment.The present invention is according to freshwater fish genome and core
Acid sequence feature is innovated in DNA extracting method and the selection of enrichment process middle probe, can simply, quickly be sieved
The microsatellite sequence in most freshwater fish whole gene groups is selected, develops substantial amounts of micro-satellite primers in a short time.Establish
Freshwater fish microsatellite DNA technical system, these molecular labelings are made to be applied to identify in fish germ plasma resource, genetic breeding is ground
Study carefully.
The content of the invention
It is an object of the invention to develop the screening technique of freshwater fish microsatellite sequence, can simply, quickly screen
The microsatellite sequence gone out in most freshwater fish whole gene groups, develops substantial amounts of micro-satellite primers in a short time.Establish light
Water fish class microsatellite DNA technical system, these molecular labelings are made to be applied to identify in fish germ plasma resource, genetic breeding is ground
Study carefully.The present invention is applied to most of freshwater fishes.
Technical scheme is as follows：
1st, fish muscle DNA extraction
By being carried out to fish DNA extraction effect on the basis of repeatedly comparing, extracting method innovate and improved, specifically
Extraction step is as follows：
(l) musculature of clip beans size, the alcohol attached on muscle is cleaned with ultra-pure water, is fully shredded, is put into 1.5mL
In centrifuge tube;
(2) 500 μ l lysate and 5 μ l Proteinase K are added into centrifuge tube, is digested at least 3 hours at 55 DEG C, every 10 points
Clock rocks from side to side once, until solution clear;
(3) sample digested is centrifuged 5 minutes（5000rpm, 4 DEG C）Precipitation is removed, takes supernatant to move in new pipe.
(4) isometric phenol is added（About 500 μ l), centrifuge tube is placed on rotation ware and slowly overturned 20 minutes, with temperature
Centrifuged 10 minutes after the mixing two-phase of sum（8000rpm, 4 DEG C）.
(5) supernatant is shifted to new pipe, adds isometric phenol chloroform（24：1）, centrifuge tube is placed on rotation ware and delayed
Slowly centrifuged 15 minutes after overturning 15 minutes（8000rpm, 4 DEG C）.
(6) supernatant is shifted to new pipe, adds isometric chloroform isoamyl alcohol（24：1）, centrifuge tube is placed in rotation ware
Centrifuged 10 minutes after above slowly overturning 15 minutes（8000rpm, 4 DEG C）.
(7) supernatant is taken to add the sodium acetate of 0.1 times of volume into new centrifuge tube（About 50 μ l) and the cold nothing of 2.5 times of volumes
Water-ethanol（About 1000 μ l) be put into -20 DEG C precipitation at least 3 hours after centrifuge 10 minutes（12000rpm, 4 DEG C）Abandon supernatant.
(8) plus after 70% ethanol elution centrifuge 10 minutes（12000rpm, 4 DEG C）After abandon supernatant.Test tube is stood upside down, in room
Temperature is lower to be spontaneously dried;
(9) dissolve:60 μ l ultra-pure water dissolving DNA is added, 4 DEG C of preservations are standby.
2nd, digestion
With the digestion genomic DNAs of Mbo I.37 degree of water-baths, digest 12-16 hours.
Digestion products electrophoresis in 2 ﹪ Ago-Gels, the gel of 300-800bpDNA fragments is cut, reclaimed with DNA pure
Change kit recovery.
It is connected with joint
Endonuclease bamhi withSau LA /Sau LB joints connect.Joint sequence is as follows
（Sau LA /Sau LB）
Oligo A：5 ＇-GGCCACAGACCCCACGCATCG-3 ＇ (SEQ ID NO.1)
With
Oligo B：The ＇ of 5 ＇-PO4- GATACGAAGCTTGGGCTCTCTCG CC- 3 (SEQ ID NO.2)
4 enrichment with magnetic bead microsatellite sequences
AGC, ACAG, ATCT is being selected to repeat sequence according to the characteristics of microsatellite repetitive sequence in freshwater fish during enrichment with magnetic bead
The probe of row is enriched with.Comprise the following steps that：
Using Dynal M-280 Streptavidins (strep tavidin) coated enrichment with magnetic bead microsatellite sequence.Experiment institute
Need reagent and material：Magnetic bead (Streptavidin magnesphere Paramagnetic Particles, Promega),
0.5 × SSC, 0.1 × SSC, 10%SDS (regulation pH to 7.5), 0.15mol/LNaOH, 1.25mol/L acetic acid, OligoA-
Sau3AL（50 pmol/μl）,-the Biotin- (AGC) of 10pmol/ μ l biotinylated probes 5 '8, 5’-Biotin-(ACAG)6,5’-
Biotin-(ATCT)6。
Initially set up the 40 μ l hybridization systems of probe and joint endonuclease bamhi：
The μ l of endonuclease bamhi 15 of jointing
Biotinylated probes（20 pmol/μl） 1μl
OligoA-Sau3AL（50 pmol/μl） 4μl
20×SSC 10μl
10%SDS（Final concentration 0.1%） 0.5μl
The μ l of high purity water 9.5
95 DEG C are denatured 5min, are incubated 1 hour at 68 DEG C.
Magnetic bead is balanced, forms suspension.Magnetic bead is captured with magnetic frame, pipe is placed on magnetic frame, magnetic bead is concentrated on pipe
Side, carefully remove supernatant.Magnetic bead is washed with 0.5 × SSC (300 μ l every time) 3 times.Every time after washing, captured with magnetic frame
Magnetic bead simultaneously carefully removes supernatant.Magnetic bead is resuspended in 200 μ l 6 × SSC (containing 0.1%SDS).Then capture probe-joint enzyme
Hybridization solution after section of cutting into slices hybridization, obtains the mononucleotide sequence repeated rich in SSR, and specific operation process is as follows：
（1）Hybridization solution is added in the magnetic bead after washing balance, react 20min at room temperature, accompany by every 1-2min upset；
（2）Magnetic bead is captured with magnetic frame and carefully removes supernatant.300 μ l 6 × SSC (containing 0.1%SDS) is added in magnetic bead,
Elute 10 minutes at room temperature；
（3）Supernatant is removed, adds at 300 μ l 68 DEG C of 6 × SSC (containing 0.1%SDS) and elutes 10 minutes, be repeated once；
（4）300 μ 6 × SSC of l room temperature elutions 10 minutes, are repeated once；
（5）200 3 × SSC of μ l are eluted 10 minutes at room temperature, are repeated once；
At this moment the NaoH that 50 μ l 0.15mol/L are added in the centrifuge tube where magnetic bead reacts 20 minutes at room temperature, make DNA from
Magnetic bead surfaces separate, and then take supernatant in a clean centrifuge tube, add 5.5 10 × TBE of μ l and 3.25 μ l
In 1.25mol/L acetic acid and NaOH, now single-stranded target SSR DNA sequence dnas are can obtain.Finally it is subject to purification kit
Purifying.
Expand the DNA fragmentation containing microsatellite sequence
Using capture dna as template, Sau3AL is that primer establishes 50 μ l double-stranded DNAs recovery PCR reaction systems, and PCR cycle program is set
It is set to：95 DEG C of pre-degenerations 3 minutes, then 5 circulations include 95 DEG C of denaturation 30s, and 53 DEG C of annealing 30s, 72 DEG C extend 45s, then
Carry out 92 DEG C of denaturation 30s, 53 DEG C of annealing 30s, 72 DEG C of extension 55s, most after extending 30 minutes at 72 DEG C of other 30 circulations.
Detection PCR primer simultaneously uses Purification Kit.
Connect carrier T, Cloning Transformation
The PCR purified products expanded are connected with pGEM-TEasy carriers (Promega), 10 are established according to support agent box explanation
μ l linked systems, overnight, connection liquid is reconstituted in competent cell DH5a, and the competent cell of oneself conversion is coated with for 4 DEG C of connections
In addition 0.5mM IPTG and 50 μ g/ml X-Gal Amp+ On LB flat boards, 37 DEG C of overnight incubations incubate, and are afterwards placed in flat board
4 DEG C of refrigerator overnights, this will be helpful to the expression of blue hickie.White colony is selected, in 200 μ l Amp+In LB fluid nutrient mediums,
37 DEG C of shaking table 4-7 hours, expand culture.
PCR methods screening containing microsatellite sequence positive colony
Colony PCR amplification system is:50ng DNA (0.5 μ L), Oligo A primers 0.5 μ L, (AGC)8Primer 0.5 μ L, 10 ×
The 1 μ L of μ L, Mg2+ of Buffer 1.5,100 μm of μ L of ol/L dNTP 1, Taq polymerase (0.05 μ L), add ultra-pure water to 15 μ L.Expand
Increase program:96 DEG C of pre-degenerations 5 minutes, 96 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 60s, most after extending 10 at 72 DEG C
Minute.
If bacterium colony PCR primer can provide the band of two or the above, we are considered as this monoclonal containing purposeful
Sequence, it is on the contrary then do not have.What we will be singled out thinks that the bacterium solution containing purposeful SSR sequences is delivered to biotech firm and is sequenced.
Sequence analysis
Using DNA star software editing sequences, the genome sequence that carrier sequence obtains first is removed.Found out and contained using SSRhunte
There is the sequence of microsatellite（Number of repetition >=5）.
Specific embodiment
By taking long-snout catfish as an example, according to " content of the invention " step operation, obtain as：
SEQ ID NO.3
SEQ ID NO.4
SEQ ID NO.5
Shown microsatellite sequence.
Brief description of the drawings
Fig. 1 is fish tissue sample DNA electricity trip figure.
Sequence table
<110>Xinyang institute
<120>A kind of method of freshwater fish screening microsatellite sequence
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
ggccacagac cccacgcatc g 21
<210> 2
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
gatacgaagc ttgggctctc tcg 23
<210> 3
<211> 822
<212> DNA
<213>Long-snout catfish (Leiocassis longirostris)
<400> 3
ttgtatacga ctcactatag ggcgaattgg gcccgacgtc gcatgctccc ggccgccatg 60
gcggccgcgg gaattcgatt ggccagagac cccaagcttc ggatcagacc atggggaagt 120
gagataaata gtctgtgtgt gtgtgtgtgg gtgtgtgtgt gtgtgtgtgt gtgtgagtgt 180
gagaaagaga aagtgagaga cagagagagg gagagagaga gagagaactt aatgaaagat 240
ggagaaaaga gggagagaca gagagacata ggtagagata tagaaaggca gagaaagtaa 300
agagaatttt tgaggggtgt gtgagagaga gagagagaga gagagagaga gagagagaga 360
gagagagcct gactccagtg tgttcagaat aaaacttgat tttatgattt acattaatgc 420
tgctataatg ctcgtgtcag aggctttcat aagctctagc tgaggattac acctctctga 480
tccgaagctt ggggtctctg gccaatcact agtgaattcg cggccgcctg caggtcgacc 540
atatgggaga gctcccaacg cgttggatgc atagcttgag tattctatag tgtcacctaa 600
atagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa 660
ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga 720
gctaaactca cattaattgc gttgcgctca ctgcccgctt tcccagtcgg gaaacctgtc 780
gtgccagctg cattaatgaa tcggccaacg cgcggggaga gg 822
<210> 4
<211> 1170
<212> DNA
<213>Long-snout catfish (Leiocassis longirostris)
<400> 4
tgcatcgggc agtgatgtat acgactcact atagggcgaa ttgggcccga cgtcgcatgc 60
tcccggccgc catggcggcc gcgggaattc gattggccag agaccccaag cttcggatca 120
ctgcatccac agcaccccca tgtgaccaac tctctctgtc tctcactcac acactcacac 180
actcacacac cagtgtggac tgagtttgca gtgctattac taagcctgta acactacagg 240
cagtgtgaca gacgaaacac agaacacaca cacactatga atcactgcac agcaccgtac 300
acacaggaca tatgagttta ggaagacgtc tgaattcgtg ttaacatgca acggattaac 360
gtgtgtcttc agctccgctt ctctctctct ctctctctct ctcacacaca cacacacaca 420
cacacacgtt tatcctatac gtattcttct ttctctctct aatttgtttc ccactcctct 480
ctctctctct ctctctctct ctctctctct ccctcataat gagacaggtt ggtgtattgg 540
ttggagacaa gacctttaca tttagaccac tggtatgatc cgaagcttgg ggtctctggc 600
caatcactag tgaattcgcg gccgcctgca ggtcgaccat atgggagagc tcccaacgcg 660
ttggatgcat agcttgagta ttctatagtg tcacctaaat agcttggcgt aatcatggtc 720
atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg 780
aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt 840
gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg 900
ccaacgcgcg gggagaggcg gtttgcgtat tggcgcttct tccgcttcct cgctcactga 960
ctcgctgcgc tcgggtcgtt cggctggcgg cgagcggtat cagcttcact tcaaaacgcg 1020
gtatacgggt tatccacaga atcaagggga taacgcagga aaagaacatg tgagcaaaag 1080
ggccagcaaa cgtccaaggg aatcgttaaa agtccgccag tggcttggcg tttattccat 1140
agtccgtctc tctgacgaag cattcacaaa 1170
<210> 5
<211> 592
<212> DNA
<213>Long-snout catfish (Leiocassis longirostris)
<400> 5
gggatcggca gtgatgtata cgactcacta tagggcgaat tgggcccgac gtcgcatgct 60
cccggccgcc atggcggccg cgggaattcg attggccaga gaccccaagc ttcggatcag 120
catgaggctg tcgctcactc ctaatgccga cggagaaatg ggcggagcta ccacaaccgg 180
ggtgagagaa ggaaggatgg gtggatgggt ctgtagatag atagagggat ggtgggtggc 240
tggtggctga agtgtatagg tgagaggact gatttggatg gagggatggt cacattcagg 300
gatggaggga taaatagata tagatgtgga tggtgaatgg aaggatggag aaagggggag 360
aggttgttgg aatggtattg tggttagagg ggatggaatg attctctctc tctctctctc 420
tctctctctc tctctctctc tctctctctc tctctcgcgg gggcgggctc gtcagtaatc 480
gagggtgcgt tcctctctac gactgaagga ttgagtgatc agtatattgc agtcactggt 540
caatcaattt tgtattattg cagattgttg gtccatcttt tataaaaaac aa 592
Claims (4)
1. a kind of method of freshwater fish screening microsatellite sequence, its step include：
（1）Fish muscle DNA extraction；
（2）With the digestion genomic DNAs of Mbo I, agarose gel electrophoresis, the gel of 300-800bpDNA fragments is cut, is reclaimed；
（3）Endonuclease bamhi is connected with Sau LA/Sau LB joints, and joint sequence is：
Oligo A：5 ＇-GGCCACAGACCCCACGCATCG-3 ＇ (SEQ ID NO.1)
With
Oligo B：The ＇ of 5 ＇-PO4- GATACGAAGCTTGGGCTCTCTCG CC- 3 (SEQ ID NO.2)；
（4）Using magnetic bead, the probe of AGC, ACAG, ATCT repetitive sequence is selected to carry out enrichment microsatellite sequence, acquisition is rich in SSR
The mononucleotide sequence repeated；
（5）Using Sau3AL as primer, DNA fragmentation of the PCR amplifications containing microsatellite sequence；
（6）The PCR purified products expanded are connected with pGEM-TEasy carriers (Promega), Cloning Transformation；
（7）PCR methods screening containing microsatellite sequence positive colony, bacterium colony PCR primer can provide the band of two or the above,
Illustrate that this monoclonal contains aim sequence, it is on the contrary then do not have；
（8）Sequence analysis, using DNA star software editing sequences, the genome sequence that carrier sequence obtains first is removed, is utilized
SSRhunte finds out the sequence containing microsatellite.
A kind of 2. method of freshwater fish screening microsatellite sequence according to claim 1, it is characterised in that step（5）
The PCR reaction conditions are：95 DEG C of pre-degenerations 3 minutes, then 5 circulations include 95 DEG C of denaturation 30s, 53 DEG C of 30s that anneal, 72 DEG C
Extend 45s, then carry out 92 DEG C of denaturation 30s, 53 DEG C of annealing 30s, 72 DEG C of extension 55s, most after 72 DEG C of other 30 circulations
Lower extension 30 minutes.
A kind of 3. method of freshwater fish screening microsatellite sequence according to claim 1, it is characterised in that step（6）
During the Cloning Transformation, white colony need to be selected, in 200 μ l Amp+ LB fluid nutrient mediums, 37 DEG C of shaking table 4-7 are small
When, expand culture.
A kind of 4. method of freshwater fish screening microsatellite sequence according to claim 1, it is characterised in that step（7）
The PCR of described microsatellite sequence positive colony, its reaction condition are：96 DEG C of pre-degenerations 5 minutes, 96 DEG C of denaturation 30s, 56 DEG C are moved back
Fiery 30s, 72 DEG C of extension 60s, most after extending 10 minutes at 72 DEG C.
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| CN109468391A (en) * | 2018-12-29 | 2019-03-15 | 中国水产科学研究院长江水产研究所 | Dual-PCR method applied to black carp Relationship iden- tification |
| CN109468391B (en) * | 2018-12-29 | 2021-05-25 | 中国水产科学研究院长江水产研究所 | Double PCR method applied to black carp genetic relationship identification |
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