CN104846110A

CN104846110A - Epinephelus moara detection primer based on microsatellite marker Epinmoyzh and method thereof

Info

Publication number: CN104846110A
Application number: CN201510291537.2A
Authority: CN
Inventors: 刘云国; 刘凌霄
Original assignee: Yantai University
Current assignee: Yantai University
Priority date: 2015-06-01
Filing date: 2015-06-01
Publication date: 2015-08-19
Anticipated expiration: 2035-06-01
Also published as: CN104846110B

Abstract

The invention discloses an epinephelus moara heritable variation detection method based on microsatellite marker Epinmoyzh. The method comprises the following steps: firstly extracting the genome DNA in the epinephelus moara blood and diluting for later use; designing specific primers on two ends of the sequence by using a microsatellite core sequence contained in the epinephelus moara DNA sequence; performing PCR amplification on the genome DNA of different geographic stocks of epinephelus moara or epinephelus moara individuals, and detecting the PCR product; analyzing the strip of the product, thereby determining the genotype of each individual and obtaining the genetic polymorphism map of epinephelus moara. By adopting the detection primer, the genetic variation map of the epinephelus moara genetic marker Epinmoyzh can be rapidly obtained; the method is simple, and the genotype of each epinephelus moara individual can be intuitively detected through the obtained result; the detection primer is applied to genetic marking of different geographic stocks of epinephelus moara or epinephelus moara individuals, the genealogical identification and the construction of the genetic map.

Description

Saladifish based on microsatellite marker Epinmoyzh detects primer and method

Technical field

The invention belongs to saladifish ( epinephelus moara) DNA molecular Genetic Markers, relate to a kind of technological method detecting saladifish microsatellite marker Epinmoyzh, specifically the detection method of saladifish microsatellite marker Epinmoyzh.

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 Epinmoyzh, 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 Epinmoyzh) 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 Epinmoyzh, and this microsatellite marker Epinmoyzh 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:

tacaatgatc atactgcacg ggcaatggaa ttcagtaagc attttagcta

cacacacaca cacacacaca cacacacaca cacacacaga acacacacac

taaaattttt aacgttaagt tgctagattt cccaggagac cagctgaaac

tgaatgcatt tgttgattac ggc

Wherein micro-satellite core sequence is: cacacacacacacacacacacacacacacacacacacaga acacacacac

Epinmoyzh micro-satellite primers sequence is:

Normal chain 5 '-TACAATGATCATACTGCACGG-3 ',

Minus strand 5 '-GCCGTAATCAACAAATGCATTC-3 ', uses annealing temperature during this primer to be 52 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, 52 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 Epinmoyzh can be obtained.

The invention has the beneficial effects as follows the mapping genetic variations of the Epinmoyzh microsatellite marker that can obtain saladifish efficiently, 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 Epinmoyzh 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 Epinmoyzh 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 '-TACAATGATCATACTGCACGG-3 ', minus strand 5 '-GCCGTAATCAACAAATGCATTC-3 ', uses annealing temperature during this primer to be 52 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, 52 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 Epinmoyzh with the glacial acetic acid solution immersion 5min of 10%, as shown in Figure 1.Fig. 1 result shows, 20 individual coamplifications of saladifish go out 7 allelotrope, illustrate that microsatellite marker Epinmoyzh has more much higher state property, be applicable to the investigation and application carrying out the aspect such as saladifish genetic diversity and genetic construction analysis, individual recognition and genealogical identification.

<110> University Of Yantai

<120> detects primer and method based on the saladifish of microsatellite marker Epinmoyzh

<160> 1

<210> 1

<211> 173

<212> DNA

<213> saladifish (Epinephelus moara)

<220>

<221> repeat_region

<222> (51)...(100)

<400> 1

tacaatgatc atactgcacg ggcaatggaa ttcagtaagc attttagcta cacacacaca 1

cacacacaca cacacacaca cacacacaga acacacacac taaaattttt aacgttaagt 61

tgctagattt cccaggagac cagctgaaac tgaatgcatt tgttgattac ggc 121

Claims

1. one group of primer detected saladifish heritable variation based on microsatellite marker Epinmoyzh, is characterized in that, primer sequence is:

Normal chain 5 '-TACAATGATCATACTGCACGG-3 ',

Minus strand 5 '-GCCGTAATCAACAAATGCATTC-3 ' is 52 DEG C by annealing temperature during this mark.

2. based on the method that microsatellite marker Epinmoyzh detects saladifish heritable variation, it is characterized in that, first extract 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 determines the genotype of saladifish Different Individual in Epinmoyzh core sequence district;

Wherein said saladifish microsatellite marker Epinmoyzh and Auele Specific Primer are:

Normal chain 5 '-TACAATGATCATACTGCACGG-3 ',

3. detection method 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, 52 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. detection method 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, soak 30min with the silver nitrate solution of 0.1% again, 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 detection method according to claim 2 between saladifish colony on genetic polymorphism map construction.