CN105755165B - A kind of the genotype detection primer and method of lefteye flounder microsatellite marker Paraoliva-2 - Google Patents
A kind of the genotype detection primer and method of lefteye flounder microsatellite marker Paraoliva-2 Download PDFInfo
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- CN105755165B CN105755165B CN201610332146.5A CN201610332146A CN105755165B CN 105755165 B CN105755165 B CN 105755165B CN 201610332146 A CN201610332146 A CN 201610332146A CN 105755165 B CN105755165 B CN 105755165B
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Abstract
The invention discloses the genotype detection primers and method of a kind of lefteye flounder microsatellite marker Paraoliva-2.The genomic DNA in Paralichthys olivaceus is extracted first and is diluted spare；The microsatellite core sequence contained in lefteye flounder DNA sequence dna is recycled, in its sequence design specific primers at both ends；Then PCR amplification is carried out using genomic DNA individual in the primer pair lefteye flounder difference proles or lefteye flounder group, PCR product is detected；It is analyzed using the band that product occurs, determines the genotype of each individual, and obtain the genetic polymorphism map of lefteye flounder.The present invention can efficiently obtain the mapping genetic variations of lefteye flounder genetic marker Paraoliva-2, and method is easy.
Description
Technical field
The invention belongs to lefteye flounder (Paralichthys olivaceus) DNA molecular Genetic Markers, it is related to a kind of inspection
Survey the technical method of lefteye flounder microsatellite marker Paraoliva-2, the specifically genotype of lefteye flounder microsatellite marker Paraoliva-2
Detection method.
Background technique
Lefteye flounder (Paralichthys olivaceus) belong to Pleuronectiformes (Pleuronectiformes), lefteye flounder section
(Paralichthyidae), Paralichthys (Paralichthys), it is referred to as flatfish (Flatfish, Fluke) in China, it is left
Flounder (Left-eyed flounder) tooth piece and step (Plaice) are the rare economic fish of marine products that cold aqueous, bottom is dwelt.At me
There is extensive distribution in the Bohai and Yellow Seas of state and the East Sea.Lefteye flounder has growth fast, and individual is big, and reproductive capacity is strong, migratory small, returns
The features such as Gui Xingqiang, particularly suitable in coastal development aquatic breeding.China artificial breeding of lefteye flounder and quotient since the eighties
Product cultivation, occupies an important position in sea-farming at present.However, overfishing in recent years has made its stock number seriously decline
Subtract, and the fast development of aquatic breeding, also the inheritance of Ya Xian natural population, germ plasm resource and genetic diversity are produced
Immeasurable influence.Molecular labeling can be applied to the side such as analysis of genetic diversity, individual identification and genealogical identification of lefteye flounder
Face.Although isoenzyme mark, RAPD label and AFLP labelling technique can be used for genetic structure and its heredity of research lefteye flounder
Diversity, however since these labels belong to dominant inheritance label, detection efficiency is not as good as codominant markers such as microsatellite markers
It is high.In addition, the shortcomings that isoenzyme mark is that polymorphism is low, the stability of RAPD label is bad, and AFLP marking operation is cumbersome, this
Their application is all greatly limited a bit.And microsatellite sequence is distributed widely in eukaryotic gene group, its main feature is that type
More, allele number is more, and polymorphism is high, codominance, mendelian inheritance pattern, and is randomly distributed in genome, Er Qiewei
Satellite markers detection efficiency is high, as a result stable.Currently, although having developed some microsatellite markers in lefteye flounder, they
The disadvantage is that allele is few, polymorphism is not high, limits its application, so there is an urgent need to obtain the microsatellite marker of high polymorphism
To carry out the research and application of lefteye flounder genetic diversity, individual identification and genealogical identification etc..
Summary of the invention
The object of the present invention is to provide a kind of lefteye flounder DNA molecular Genetic Markers of high polymorphism, i.e. lefteye flounder microsatellite
The rapid detection method of Paraoliva-2 is marked, to make up the deficiency of prior art.
Basic conception of the invention mainly utilizes the microsatellite core sequence contained in lefteye flounder DNA sequence dna, at its both ends
Design specific primer simultaneously carries out PCR detection, thus the rapidly each individual hereditary variation in this microsatellite area of detection lefteye flounder,
The primer (microsatellite marker Paraoliva-2) is obtained to the polymorphism map of lefteye flounder, is intuitively detected by map each
The genotype of individual.Status and actual requirement based on the above background, the present invention obtain a microsatellite from lefteye flounder DNA sequence dna
Label, is named as Paraoliva-2, microsatellite marker Paraoliva-2 can be used for lefteye flounder analysis of genetic diversity and pedigree
Certification.
The present invention is completed according to following operational aspect: the genomic DNA and dilute first in extraction Paralichthys olivaceus
It releases spare；The microsatellite core sequence contained in lefteye flounder genomic dna sequence is recycled, design specificity is drawn at its sequence both ends
Object；Then PCR amplification is carried out using genomic DNA individual in the primer pair lefteye flounder difference proles or lefteye flounder group, PCR is produced
Object carries out denaturing polyacrylamide gel electrophoresis detection, so that it is determined that the genotype of each individual, obtains the genetic polymorphism of lefteye flounder
Property map.
Lefteye flounder genomic DNA is extracted, 100ng/ μ l is diluted to, 1 μ l is added in each PCR reaction, reacts total volume
For 25 μ l.
DNA sequence dna containing microsatellite sequence are as follows:
1 ccacatgtat gtatttgtca gggagtagta ggtttgctta cattcaatac aaacaatatg
61 atagttttat gtttgttgtt acaactacac cacacacaca cacacacaca cacacacaca
121 cacacacaca cacacacaca cacacacaca cacacacaca cacacacaca cacacacaca
181 cacacacaca cacacacaca gggatccaaa tacacaagaa gggcaacact acccctgaag
241 tatgttgggc aatacgctgc agagctgcac gatatttaaa tttagctggg acatcttctg
301 ctcggc
Wherein microsatellite core sequence are as follows:
cacacacaca cacacacaca cacacacaca cacacacaca cacacacaca cacacacaca
cacacacaca cacacacaca cacacacaca cacacacaca cacacacaca
Paraoliva-2 micro-satellite primers sequence are as follows:
Normal chain 5 '-CCACATGTATGTATTTGTCAGGGAG -3 ',
Minus strand 5 '-GCCGAGCAGGAAGATGTCCCAGCT -3 ', annealing temperature when using the primer are 60 DEG C.
Microsatellite core sequence and primer sequence are core of the invention, are presented in lefteye flounder Diversity Detection polymorphic
Property.
The sample-adding parameter of PCR amplification are as follows: each PCR reaction total volume is 25 μ l, including 100ng lefteye flounder genomic DNA；10
× PCR Buffer, 2.5 μ l；Mg2+1.5mmol/L；Tag enzyme 1u；Each 0.1mmol/L of dNTP；Each 10pmol of above-mentioned primer；
Add ddH2O to 25 μ l.Use the program parameter that PCR instrument is set when the primer are as follows: 94 DEG C of denaturation 2min；94 DEG C of 30sec, 60 DEG C
45sec, 72 DEG C of 40sec, the reaction carry out 35 circulations；72 DEG C of 5 min of extension, 4 DEG C of preservations.
The detecting step of PCR product: by PCR product with 15W invariable power electrophoresis in 8% denaturing polyacrylamide gel
It is separated within 1.5 hours.After electrophoresis, 30min is impregnated with 10% glacial acetic acid solution first, then distilled water flushing
5min, then with 0.1% silver nitrate solution impregnate 30min, with 3% sodium carbonate liquor develop the color 5min, finally with 10% ice
Acetum impregnates 5min, and the polymorphism map of lefteye flounder microsatellite marker Paraoliva-2 can be obtained.
Therefore, it is characteristic of the invention that the hereditary variation of the Paraoliva-2 microsatellite marker of lefteye flounder can be obtained efficiently
Map, method are easy.Moreover, microsatellite marker meets Mendelian inheritance mould for other molecular genetic marker techniques
Formula, is in codominant inheritance, and acquired results can intuitively detect the genotype of each individual of lefteye flounder；And it is applied to different proles
Or genetic marker, genealogical identification and building of genetic map etc. between individual in lefteye flounder group.
Detailed description of the invention
Fig. 1 is microsatellite marker Paraoliva-2 of the present invention, and to lefteye flounder 20 individual test maps, (number 1-20 is
20 individuals of lefteye flounder).
Specific embodiment
Describe lefteye flounder Paraoliva-2 microsatellite core sequence DNA molecular genetic marker skill in detail below by embodiment
Art method.The genomic DNA in Paralichthys olivaceus is extracted first and is diluted spare；Recycle what is contained in lefteye flounder DNA sequence dna micro- to defend
Star core sequence, in its sequence design specific primers at both ends；Then the primer pair lefteye flounder difference proles or lefteye flounder group are used
The genomic DNA of interior individual carries out PCR amplification, carries out denaturing polyacrylamide gel electrophoresis detection to PCR product, determines each
The genotype of individual obtains the genetic polymorphism map of lefteye flounder.
1, the extraction of lefteye flounder genomic DNA: 100 μ g Paralichthys olivaceus are added in Eppendorf pipe, cell cracking is added
The 5 μ l of Proteinase K of liquid 500 μ l and 20mg/ml, gently shake up, and 37 DEG C overnight.Then 600 μ are added in cracked sample
L Fen Lv ︰ Fang ︰ isoamyl alcohol (25 ︰, 24 ︰ 1) mixed liquor, 12000rpm is centrifuged 10min after shaking 20min, takes supernatant.It adds
Fen ︰ Lv Fang ︰ isoamyl alcohol mixed liquor repeats aforesaid operations 3 times.Supernatant is taken again, and the cooling dehydrated alcohol and 1/ of 2 times of volumes is added
The sodium acetate of the 3mol/L of 10 volumes, 12000rpm are centrifuged 10min, discard supernatant liquid, retain DNA precipitating, then with 70% ethyl alcohol
It washs the precipitating 2 times, after ethyl alcohol volatilization completely, with TE buffer solution DNA, and is diluted to 100ng/ μ l, 4 DEG C of preservations.
2, the design of micro-satellite primers: on the basis of lefteye flounder DNA sequence dna, using the sequence of microsatellite DNA two sides same
Species are relative to the well-conserved of core sequence, accordingly in its design specific primers at both ends, amplify the site with it
Microsatellite segment.Since microsatellite core sequence mutation rate is relatively high, the increasing of microsatellite DNA core sequence number of repetition is caused
It adds deduct few, i.e. the variation of microsatellite DNA sequence length, this is the root for detecting microsatellite polymorphism.Microsatellite of the invention
The specific primer sequence at area core sequence both ends are as follows: normal chain 5 '-CCACATGTATGTATTTGTCAGGGAG -3 ', minus strand
5 '-GCCGAGCAGGAAGATGTCCCAGCT -3 ', annealing temperature when using the primer are 60 DEG C.
3, PCR amplification: being loaded first, and sample-adding amount is as follows: lefteye flounder genomic DNA (100ng/L), 1 μ l；10×PCR
Buffer, 2.5 μ l；Mg2+(25mmo1/L), 1.5 μ l；Taq enzyme (5u/ μ l), 0.2 μ l；DNTP (each 2.5mmo/L), 1 μ l；Draw
Object (each 10pmol/ μ l), 1 μ l；Add aqua sterilisa to 25 μ l.Secondly, carrying out PCR reaction, PCR amplification instrument program parameter are as follows: 94
DEG C denaturation 2min；94 DEG C of 30sec, 60 DEG C of 45sec, 72 DEG C of 40sec, 35 circulations；72 DEG C of extension 5min, 4 DEG C of preservations.
4, the detection of PCR product: PCR product is separated by electrophoresis on 8% denaturing polyacrylamide gel, electrophoresis apparatus function
Rate is 15W, and electrophoresis time is 1.5 hours or so.After electrophoresis, 30min is impregnated with 10% glacial acetic acid solution first, then
Distilled water flushing 5min, 0.1% silver nitrate solution impregnate 30min, 3% sodium carbonate liquor colour developing 5min, finally with 10%
Glacial acetic acid solution impregnate 5min lefteye flounder can be obtained in the polymorphism map of Paraoliva-2 microsatellite core sequence, such as Fig. 1
It is shown.Fig. 1 the result shows that, 20 of lefteye flounder individual coamplifications go out 23 allele, illustrate microsatellite marker Paraoliva-2
With very high polymorphism, it is appropriate for the side such as lefteye flounder genetic diversity and genetic structure analysis, individual identification and genealogical identification
The research and application in face.
<110>University Of Yantai
<120>a kind of the genotype detection primer and method of lefteye flounder microsatellite marker Paraoliva-2
<160> 1
<210> 1
<211> 306
<212> DNA
<213>lefteye flounder (Paralichthys olivaceus)
<220>
<221> repeat_region
<222> (91)...(200)
<400> 1
1 ccacatgtat gtatttgtca gggagtagta ggtttgctta cattcaatac aaacaatatg
61 atagttttat gtttgttgtt acaactacac cacacacaca cacacacaca cacacacaca
121 cacacacaca cacacacaca cacacacaca cacacacaca cacacacaca cacacacaca
181 cacacacaca cacacacaca gggatccaaa tacacaagaa gggcaacact acccctgaag
241 tatgttgggc aatacgctgc agagctgcac gatatttaaa tttagctggg acatcttctg
301 ctcggc
Claims (4)
1. the method detected using microsatellite marker Paraoliva-2 to lefteye flounder genotype, which is characterized in that extract first
Genomic DNA in Paralichthys olivaceus simultaneously dilutes spare；The microsatellite core sequence contained in lefteye flounder DNA sequence dna is recycled, is used
Specific primer carries out PCR amplification to the genomic DNA of lefteye flounder Different Individual, and it is solidifying to carry out denaturing polyacrylamide to PCR product
Gel electrophoresis detection, so that it is determined that genotype of the lefteye flounder Different Individual in Paraoliva-2 core sequence area, wherein the lefteye flounder
The specific primer of microsatellite marker Paraoliva-2 are as follows:
Normal chain 5 '-CCACATGTATGTATTTGTCAGGGAG -3 ',
Minus strand 5 '-GCCGAGCAGGAAGATGTCCCAGCT -3 ', annealing temperature when using the primer are 60 DEG C；It is wherein micro-
The core sequence of satellite markers Paraoliva-2: cacacacaca cacacacaca cacacacaca cacacacaca
cacacacaca cacacacaca cacacacaca cacacacaca cacacacaca cacacacaca cacacacaca。
2. the method that lefteye flounder genotype is detected using microsatellite marker Paraoliva-2 as described in claim 1,
It is characterized in that, being loaded parameter to the PCR amplification that the genomic DNA of different lefteye flounder individuals carries out are as follows: each PCR reaction is total
Volume is 25 μ l, including 100ng lefteye flounder genomic DNA；10 × PCR Buffer, 2.5 μ l；Mg2+1.5 mmol/L；Taq enzyme
1u；dNTP 0.1mmol/L；Each 10 pmol of specific primer in claim 1；Finally plus ddH2O to 25 μ l；PCR is set
The program parameter of instrument are as follows: 94 DEG C of denaturation 2min；94 DEG C of 30sec, 60 DEG C of 45sec, 72 DEG C of 40sec, the reaction carry out 35 and follow
Ring；72 DEG C of 5 min of extension, 4 DEG C of preservations.
3. the method that lefteye flounder genotype is detected using microsatellite marker Paraoliva-2 as described in claim 1,
It is characterized in that, the step of carrying out denaturing polyacrylamide gel electrophoresis detection to PCR product: the denaturation by PCR product 8%
It is separated within 1.5 hours in polyacrylamide gel with 15W invariable power electrophoresis, 30min is impregnated with 10% glacial acetic acid solution,
Then distilled water flushing 5min, then with 0.1% silver nitrate solution impregnate 30min, with 3% sodium carbonate liquor develop the color 5min,
Finally 5min is impregnated with 10% glacial acetic acid solution.
4. application of the method between lefteye flounder group on genetic polymorphism map construction described in claim 1-3 any one.
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| CN101911918A (en) * | 2010-03-18 | 2010-12-15 | 中国水产科学研究院黄海水产研究所 | Selective breeding method for disease-resistant superior strains of paralichthys olivaceus |
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| 牙鲆3个养殖群体遗传结构的微卫星分析;邵长伟等;《渔业科学进展》;20090228;第30卷(第1期);第1.1-2.2部分 * |
| 牙鲆养殖群体遗传变异的微卫星标记研究;刘云国等;《海洋水产研究》;20051031;第26卷(第5期);第1.1-2.2部分，第32页第2段 * |
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