CN104894257A - Primer and method for detecting epinephelus radiatus by use of microsatellite marker Epinmoyn - Google Patents
Primer and method for detecting epinephelus radiatus by use of microsatellite marker Epinmoyn Download PDFInfo
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- CN104894257A CN104894257A CN201510291565.4A CN201510291565A CN104894257A CN 104894257 A CN104894257 A CN 104894257A CN 201510291565 A CN201510291565 A CN 201510291565A CN 104894257 A CN104894257 A CN 104894257A
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Abstract
The invention discloses a primer and method for detecting the genotype of epinephelus radiatus by use of a microsatellite marker Epinmoyn. The method comprises the following steps: extracting the genome DNA in the blood of epinephelus radiatus and diluting for later use; designing a specific primer by use of the microsatellite core sequence contained in the DNA sequence of epinephelus radiatus; performing PCR amplification on the genome DNA of the individuals in different geographic stocks of epinephelus radiatus or epinephelus radiatus stock by use of the primer, and detecting the PCR product; and analyzing by use of the stripes of the product to determine the genotype of each individual and obtain the genetic polymorphism map of epinephelus radiatus. According to the invention, the genetic variation map of the genetic marker Epinmoyn of epinephelus radiatus can be quickly obtained, the method is simple and convenient, and the obtained result can be used for visually detecting the genotype of each individual of epinephelus radiatus; and the method is applied to the genetic marking, genealogical identification, establishment of the genetic map and the like among the individuals of different geographic stocks or epinephelus radiatus stock.
Description
Technical field
The invention belongs to saladifish (
epinephelus moara) DNA molecular Genetic Markers, relate to a kind of technological method detecting saladifish microsatellite marker Epinmoyn, specifically the detection method of saladifish microsatellite marker Epinmoyn.
Background technology
Saladifish (
epinephelus moara) belong to Perciformes, Sushi section, Epinephelus, be commonly called as careless spot, oil mark, be distributed in North Western Pacific, the East Sea and the South Sea are originated in China.Its meat flavour is delicious, and smooth in taste, protein content is high, nutritious, deeply favors by consumers in general.Saladifish growth is fast, strong adaptability, economic worth are high, be adapted to pond, net cage and indoor industrially-cultured.But saladifish natural resources amount is few, be difficult to the demand meeting people.2010; Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science, Fujian Prov. Inst. of Aquatic Products and Laizhou, Yantai City Ming Bo aquatic products company research cooperation; obtain propagation in scale success in northern China first, carry out Grouper cultivating for the north and established solid basis.Saladifish has become China's one of the most potential sea farming new variety at present.Saladifish is as a kind of emerging breed variety, and genetics research is relatively weak, does not also have the report that microsatellite marker is developed.Although isoenzyme mark, RAPD mark and AFLP labeling technique also may be used for genetic construction and the genetic diversity thereof of studying saladifish, but mark because these marks all belong to dominant inheritance, detection efficiency is high not as codominant markers such as microsatellite markers.In addition, the shortcoming of isoenzyme mark is that polymorphism is low, and the stability of RAPD mark is bad, and AFLP marking operation is loaded down with trivial details, and these all greatly limit their application.And microsatellite sequence is distributed widely in eukaryotic gene group, be characterized in that kind is many, allelotrope number is many, and polymorphism is high, codominance, mendelian inheritance pattern, and is randomly distributed in genome, and microsatellite marker detection efficiency is high, and result is stablized.But owing to lacking saladifish microsatellite marker, limit the development of its molecule genetics research, so in the urgent need to obtaining microsatellite marker to carry out the investigation and application of the aspects such as saladifish genetic diversity, individual recognition and genealogical identification.
Summary of the invention
The object of this invention is to provide a kind of saladifish DNA molecular Genetic Markers of high polymorphism, i.e. the method for quick of saladifish microsatellite marker Epinmoyn, to make up the deficiency of prior art.
The present invention mainly utilizes the micro-satellite core sequence contained in saladifish DNA sequence dna, its design specific primers at both ends go forward side by side performing PCR detect, thus detect the heritable variation of each individuality of saladifish in this micro-satellite district rapidly, obtain this primer (microsatellite marker Epinmoyn) to the polymorphism collection of illustrative plates of saladifish, detected the genotype of each individuality by collection of illustrative plates intuitively.Based on above-mentioned background present situation and actual requirement, the present invention obtains a microsatellite marker from saladifish DNA sequence dna, called after Epinmoyn, and this microsatellite marker Epinmoyn can be used for saladifish analysis of genetic diversity and genealogical identification.
The present invention completes according to following operational aspect: first extract the genomic dna in saladifish blood and diluted for use; The micro-satellite core sequence contained in recycling saladifish genomic dna sequence, in its sequence design specific primers at both ends; Then individual genomic dna in the different proles of this primer pair saladifish or saladifish group is used to carry out pcr amplification, denaturing polyacrylamide gel electrophoresis detection is carried out to PCR primer, thus determine the genotype of each individuality, obtain the genetic polymorphism collection of illustrative plates of saladifish.
Extract saladifish genomic dna, diluted for 100ng/ μ l, add 1 μ l in each PCR reaction, reaction cumulative volume is 25 μ l.
DNA sequence dna containing microsatellite sequence is:
ccaagagcat catatcgtgc gaccctcagc aactttttaa atgtgtgtgt
gtgtgtgtgt gtgtgtgtgt gtgtgtgtat atatatatat atatatatat
gcgagggagc aattgtgagg tgctagactg gaggccagat atacccgggc
tgtgtaagtg gcggcta
Wherein micro-satellite core sequence is: gtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtat atatatatat atatatatat
Epinmoyn micro-satellite primers sequence is:
Normal chain 5 '-CCAAGAGCATCATATCGTGC-3 ',
Minus strand 5 '-TAGCCGCCACTTACACAGCC-3 ', uses annealing temperature during this primer to be 53 DEG C.
Micro-satellite core sequence and primer sequence are cores of the present invention, in saladifish Diversity Detection, present polymorphism.
The application of sample parameter of pcr amplification is: it is 25 μ l that each PCR reacts cumulative volume, comprises 100ng saladifish genomic dna; 10 × PCR Buffer, 2.5 μ l; Mg
2+1.5mmol/L; Tag enzyme 1u; The each 0.1mmol/L of dNTP; The each 10pmol of above-mentioned primer; Add ddH
2o to 25 μ l.The program parameter that PCR instrument is set during this primer is used to be: 94 DEG C of sex change 2min; 94 DEG C of 30sec, 53 DEG C of 45sec, 72 DEG C of 40sec, 35 circulations are carried out in this reaction; 72 DEG C extend 5 min, 4 DEG C of preservations.
The detecting step of PCR primer: PCR primer is separated with 15W invariable power electrophoresis in the denaturing polyacrylamide gel of 8% for 1.5 hours.After electrophoresis terminates, first 30min is soaked with the glacial acetic acid solution of 10%, then distilled water flushing 5min, 30min is soaked again with the silver nitrate solution of 0.1%, with the sodium carbonate solution colour developing 5min of 3%, finally soak 5min with the glacial acetic acid solution of 10%, the polymorphism collection of illustrative plates of saladifish microsatellite marker Epinmoyn can be obtained.
Beneficial effect of the present invention:
The present invention can obtain the mapping genetic variations of the Epinmoyn microsatellite marker of saladifish efficiently, and method is easy.And for other molecular genetic marker technique, microsatellite marker meets Mendelian inheritance pattern, in codominant inheritance, acquired results can detect the genotype of each individuality of saladifish intuitively; And be applied to the structure etc. of genetic marker, genealogical identification and genetic map between individuality in different proles or saladifish group.
Accompanying drawing explanation
Fig. 1 is the microsatellite marker Epinmoyn of the present invention detection collection of illustrative plates (numbering 1-20 are 20 individualities of saladifish) individual to saladifish 20.
Embodiment
The present invention is described in detail in saladifish Epinmoyn micro-satellite core sequence DNA molecular Genetic Markers method below by embodiment.First the genomic dna in saladifish blood is extracted and diluted for use; The micro-satellite core sequence contained in recycling saladifish DNA sequence dna, in its sequence design specific primers at both ends; Then individual genomic dna in the different proles of this primer pair saladifish or saladifish group is used to carry out pcr amplification, denaturing polyacrylamide gel electrophoresis detection is carried out to PCR primer, determine the genotype of each individuality, namely obtain the genetic polymorphism collection of illustrative plates of saladifish.
1, the extraction of saladifish genomic dna: 100 μ l saladifish blood are added in Eppendorf pipe, then add the Proteinase K 5 μ l of cell pyrolysis liquid 500 μ l and 20mg/ml, shake up gently, 37 DEG C are spent the night.Then in cracked sample, add 600 μ l Fen Lv ︰ Fang ︰ primary isoamyl alcohol (25 ︰ 24 ︰ 1) mixed solutions, rock the centrifugal 10min of 12000rpm after 20min, get supernatant liquor.Add Fen ︰ Lv Fang ︰ primary isoamyl alcohol mixed solution again, repeat aforesaid operations 3 times.Get supernatant liquor again, add the sodium-acetate of the dehydrated alcohol of 2 times of volume coolings and the 3mol/L of 1/10 volume, the centrifugal 10min of 12000rpm, abandoning supernatant, retains DNA precipitation, then uses this precipitation of 70% washing with alcohol 2 times, after ethanol volatilization completely, with TE buffer solution DNA, and dilution is 100ng/ μ l, 4 DEG C of preservations.
2, the design of micro-satellite primers: on saladifish DNA sequence dna basis, utilize the sequence of microsatellite DNA both sides well-conserved relative to core sequence of same species, accordingly in its design specific primers at both ends, amplify micro-satellite fragment in this site with it.Because micro-satellite core sequence mutation rate is relatively high, cause increase or the minimizing of microsatellite DNA core sequence multiplicity, i.e. the change of microsatellite DNA sequence length, this is the root detecting microsatellite polymorphism.The specific primer sequence at core sequence two ends, micro-satellite district of the present invention is: normal chain 5 '-CCAAGAGCATCATATCGTGC-3 ', minus strand 5 '-TAGCCGCCACTTACACAGCC-3 ', uses annealing temperature during this primer to be 53 DEG C.
3, pcr amplification: first application of sample, application of sample amount is as follows: saladifish genomic dna (100ng/L), 1 μ l; 10 × PCR Buffer, 2.5 μ l; Mg
2+(25mmo1/L), 1.5 μ l; Taq enzyme (5u/ μ l), 0.2 μ l; DNTP (each 2.5mmo/L), 1 μ l; Primer (each 10pmol/ μ l), 1 μ l; Add aqua sterilisa to 25 μ l.Secondly, carry out PCR reaction, its PCR amplification instrument program parameter is: 94 DEG C of sex change 2min; 94 DEG C of 30sec, 53 DEG C of 45sec, 72 DEG C of 40sec, 35 circulations; 72 DEG C extend 5min, 4 DEG C of preservations.
4, the detection of PCR primer: by PCR primer electrophoretic separation on the denaturing polyacrylamide gel of 8%, electrophoresis apparatus power is 15W, and electrophoresis time is 1.5 hours.After electrophoresis terminates, first 30min is soaked with the glacial acetic acid solution of 10%, then distilled water flushing 5min, the silver nitrate solution of 0.1% soaks 30min, the sodium carbonate solution colour developing 5min of 3%, finally can obtain the polymorphism collection of illustrative plates of saladifish at the micro-satellite core sequence of Epinmoyn with the glacial acetic acid solution immersion 5min of 10%, as shown in Figure 1.Result shows, 20 individual coamplifications of saladifish go out 8 allelotrope, illustrate that microsatellite marker Epinmoyn has more much higher state property, be applicable to the investigation and application carrying out the aspects such as saladifish Genetic diversity evaluation, individual recognition and genealogical identification.
<110> University Of Yantai
The primer that <120> utilizes microsatellite marker Epinmoyn to detect saladifish and method
<160> 1
<210> 1
<211> 167
<212> DNA
<213> saladifish (Epinephelus moara)
<220>
<221> repeat_region
<222> (43)...(100)
<400> 1
ccaagagcat catatcgtgc gaccctcagc aactttttaa atgtgtgtgt gtgtgtgtgt 1
gtgtgtgtgt gtgtgtgtat atatatatat atatatatat gcgagggagc aattgtgagg 61
tgctagactg gaggccagat atacccgggc tgtgtaagtg gcggcta 121
Claims (5)
1. one group of primer utilizing microsatellite marker Epinmoyn to detect saladifish genotype, is characterized in that,
Wherein said primer is sequence:
Normal chain 5 '-CCAAGAGCATCATATCGTGC-3 ',
Minus strand 5 '-TAGCCGCCACTTACACAGCC-3 ' is 53 DEG C by annealing temperature during this mark.
2. the method utilizing microsatellite marker Epinmoyn to detect saladifish genotype, is characterized in that, first extracts the genomic dna in saladifish blood and diluted for use; The micro-satellite core sequence contained in recycling saladifish DNA sequence dna, Auele Specific Primer is used to carry out pcr amplification to the genomic dna of saladifish Different Individual, denaturing polyacrylamide gel electrophoresis detection is carried out to PCR primer, thus determine the genotype of saladifish Different Individual in Epinmoyn core sequence district, wherein said saladifish microsatellite marker Epinmoyn and Auele Specific Primer are:
Normal chain 5 '-CCAAGAGCATCATATCGTGC-3 ',
Minus strand 5 '-TAGCCGCCACTTACACAGCC-3 ' is 53 DEG C by annealing temperature during this mark.
3. utilize the method that microsatellite marker Epinmoyn detects saladifish genotype as claimed in claim 2, it is characterized in that, to the pcr amplification that the genomic dna of different saladifish individuality carries out, its application of sample parameter is: it is 25 μ l that each PCR reacts cumulative volume, comprises 100ng saladifish genomic dna; 10 × PCR Buffer, 2.5 μ l; Mg
2+1.5 mmol/L; Taq enzyme 1u; DNTP 0.1mmol/L; Each 10 pmol of Auele Specific Primer in claim 1; Finally add ddH
2o to 25 μ l; The program parameter arranging PCR instrument is: 94 DEG C of sex change 5min; 94 DEG C of 45sec, 53 DEG C of 1min, 72 DEG C of 40sec, 35 circulations are carried out in this reaction; 72 DEG C extend 5 min, 4 DEG C of preservations.
4. utilize the method that microsatellite marker Epinmoyn detects saladifish genotype as claimed in claim 2, it is characterized in that, PCR primer is carried out to the step of denaturing polyacrylamide gel electrophoresis detection: PCR primer within 1.5 hours, be separated with 15W invariable power electrophoresis in the denaturing polyacrylamide gel of 8%, glacial acetic acid solution with 10% soaks 30min, then distilled water flushing 5min, 30min is soaked again with the silver nitrate solution of 0.1%, with the sodium carbonate solution colour developing 5min of 3%, finally soak 5min with the glacial acetic acid solution of 10%.
5. the application of the method described in claim 2-4 between saladifish colony on genetic polymorphism map construction.
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Non-Patent Citations (3)
| Title |
|---|
| J.-H. KANG ET AL.: "Development of microsatellite markers for the kelp grouper Epinephelus bruneus by 454 pyrosequencing and transfer to related species", 《GENETICS AND MOLECULAR RESEARCH》 * |
| 尹绍武等: "石斑鱼遗传多样性的研究进展", 《水产科学》 * |
| 赵丽丽: "3种石斑鱼微卫星标记的开发及跨种扩增", 《中国优秀硕士学位论文全文数据库》 * |
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