CN104894257B - Utilize the microsatellite marker Epinmoyn primers detected to saladifish genotype and method - Google Patents

Utilize the microsatellite marker Epinmoyn primers detected to saladifish genotype and method Download PDF

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CN104894257B
CN104894257B CN201510291565.4A CN201510291565A CN104894257B CN 104894257 B CN104894257 B CN 104894257B CN 201510291565 A CN201510291565 A CN 201510291565A CN 104894257 B CN104894257 B CN 104894257B
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saladifish
primer
epinmoyn
pcr
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CN104894257A (en
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刘云国
刘凌霄
李瑶瑶
高永林
李彦伸
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Yantai University
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Abstract

The invention discloses a kind of primer detected using microsatellite marker Epinmoyn to saladifish genotype and method.The genomic DNA in saladifish blood is extracted first and is diluted standby；Recycle the microsatellite core sequence design specific primer contained in saladifish DNA sequence dna；Then enter performing PCR using genomic DNA individual in the primer pair saladifish difference proles or saladifish group to expand, PCR primer is detected；The band occurred using product is analyzed, it is determined that the genotype of each individual, and obtain the genetic polymorphism collection of illustrative plates of saladifish.The present invention can efficiently obtain saladifish genetic marker Epinmoyn mapping genetic variations, and method is easy, and acquired results can intuitively detect the genotype of each individual of saladifish；Applied to structure of genetic marker, genealogical identification and genetic map etc. between individual in different proles or saladifish group.

Description

Drawn using microsatellite marker Epinmoyn to what saladifish genotype was detected Thing and method
Technical field
The invention belongs to saladifish（Epinephelus moara）DNA molecular Genetic Markers, it is related to a kind of inspection Saladifish microsatellite marker Epinmoyn technical method is surveyed, specifically saladifish microsatellite marker Epinmoyn's Detection method.
Background technology
Saladifish（Epinephelus moara）Belong to Perciformes, Sushi sections, Epinephelus, be commonly called as careless spot, oil mark, point North Western Pacific is distributed in, the East Sea and the South Sea are originated in China.Its meat flavour is delicious, smooth in taste, and protein content is high, and nutrition is rich Richness, it is deep to be favored by consumers in general.Saladifish growth is fast, strong adaptability, economic value are high, be adapted to pond, net cage and It is indoor industrially-cultured.But saladifish natural resources amount is few, it is difficult to meets the needs of people.2010, China Water obstetrics Huanghai Sea aquatic products research institute of research institute, Fujian Prov. Inst. of Aquatic Products and Yantai City Laizhou Ming Bo aquatic products company research cooperation are learned, first Propagation in scale success is obtained in northern China, carries out Grouper cultivating for the north and has established solid foundation.Saladifish China's one of most potential sea-farming new varieties are at present turned into.Saladifish is as a kind of emerging cultivation product Kind, genetics research is relatively weak, also the report without microsatellite marker exploitation.Although isoenzyme mark, RAPD mark and AFLP labelling techniques can be used for studying the genetic structure and its genetic diversity of saladifish, yet with these marks Dominant inheritance mark is belonged to, detection efficiency is high not as codominant markers such as microsatellite markers.In addition, the shortcomings that isoenzyme mark It is that polymorphism is low, the stability of RAPD marks is bad, and AFLP marking operations are cumbersome, and these all greatly limit their application. And microsatellite sequence is distributed widely in eukaryotic gene group, it is characterized in that species is more, allele number is more, polymorphism Height, codominance, mendelian inheritance pattern, and be randomly distributed in genome, and microsatellite marker detection efficiency is high, as a result surely It is fixed.But due to lacking saladifish microsatellite marker, the development of its molecule genetics research is limited, so there is an urgent need to obtain Microsatellite marker is taken to carry out the research of saladifish genetic diversity, individual identification and genealogical identification etc. and application.
The content of the invention
It is an object of the invention to provide a kind of saladifish DNA molecular Genetic Markers of high polymorphism, i.e. moire Grouper microsatellite marker Epinmoyn quick determination method, to make up the deficiency of prior art.
The present invention mainly utilizes the microsatellite core sequence contained in saladifish DNA sequence dna, is designed at its both ends Specific primer is gone forward side by side performing PCR detection, is become so as to rapidly detect heredity of each individual of saladifish in this microsatellite area It is different, obtain the primer（Microsatellite marker Epinmoyn）To the polymorphism collection of illustrative plates of saladifish, intuitively detected by collection of illustrative plates Go out each individual genotype.Based on above-mentioned background present situation and actual requirement, the present invention obtains from saladifish DNA sequence dna One microsatellite marker, is named as Epinmoyn, microsatellite marker Epinmoyn can be used for saladifish genetic diversity Property analysis and genealogical identification.
The present invention is completed according to following operational aspect：The genome in saladifish blood is extracted first DNA simultaneously dilutes standby；The microsatellite core sequence contained in saladifish genomic dna sequence is recycled, in its sequence two End design specific primer；Then using base individual in the primer pair saladifish difference proles or saladifish group Because a group DNA enters performing PCR amplification, denaturing polyacrylamide gel electrophoresis detection is carried out to PCR primer, so that it is determined that each individual Genotype, obtain the genetic polymorphism collection of illustrative plates of saladifish.
Saladifish genomic DNA is extracted, 100ng/ μ l is diluted to, adds 1 μ l in each PCR reactions, react 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 microsatellite core sequence is：gtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtat atatatatat atatatatat
Epinmoyn micro-satellite primers sequences are：
- the CCAAGAGCATCATATCGTGC -3 ' of normal chain 5 ',
- the TAGCCGCCACTTACACAGCC -3 ' of minus strand 5 ', the use of the annealing temperature during primer it is 53 DEG C.
Microsatellite core sequence and primer sequence are the cores of the present invention, are in saladifish Diversity Detection Existing polymorphism.
PCR amplification sample-adding parameter be：Each PCR reactions cumulative volume is 25 μ l, including 100ng saladifish genomes DNA；10 × PCR Buffer, 2.5 μ l；Mg2+1.5mmol/L；Taq enzyme 1u；Each 0.1mmol/L of dNTP；Above-mentioned primer is each 10pmol；Add ddH2O to 25 μ l.Using during the primer set PCR instrument program parameter be：94 DEG C of denaturation 2min；94℃ 30sec, 53 DEG C of 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 primer：By PCR primer in 8% denaturing polyacrylamide gel with 15W invariable power electrophoresis Separated within 1.5 hours.After electrophoresis terminates, 30min is soaked with 10% glacial acetic acid solution first, then distilled water flushing 5min, then with 0.1% silver nitrate solution soak 30min, with 3% sodium carbonate liquor develop the color 5min, finally with 10% ice Acetum soaks 5min, you can obtains saladifish microsatellite marker Epinmoyn polymorphism collection of illustrative plates.
Beneficial effects of the present invention：
The present invention can efficiently obtain the mapping genetic variations of the Epinmoyn microsatellite markers of saladifish, method letter Just.Moreover, for other molecular genetic marker techniques, microsatellite marker meets Mendelian inheritance pattern, in codominance Heredity, acquired results can intuitively detect the genotype of each individual of saladifish；And it is applied to different proles or cloud Structure of genetic marker, genealogical identification and genetic map etc. between individual in hamlet group.
Brief description of the drawings
Fig. 1 detection collection of illustrative plates that to be microsatellite marker Epinmoyn of the present invention individual to saladifish 20 (numbering 1- 20 be 20 individuals of saladifish).
Embodiment
The present invention is described in detail below by embodiment in saladifish Epinmoyn microsatellite core sequence DNA moleculars Genetic Markers method.The genomic DNA in saladifish blood is extracted first and is diluted standby；Recycle moire lithosporic The microsatellite core sequence contained in fish DNA sequence dna, in its sequence design specific primers at both ends；Then the primer pair cloud is used Individual genomic DNA enters performing PCR amplification in hamlet difference proles or saladifish group, and PCR primer is become Property polyacrylamide gel electrophoresis detection, it is determined that the genotype of each individual, that is, obtain the genetic polymorphism figure of saladifish Spectrum.
1st, the extraction of saladifish genomic DNA：100 μ l saladifishs blood are added in Eppendorf pipes, then Cell pyrolysis liquid 500 μ l and 20mg/ml the μ l of Proteinase K 5 are added, are gently shaken up, 37 DEG C overnight.Then in cracked sample 600 μ l Fen Lv ︰ Fang ︰ isoamyl alcohol are added in product（The ︰ 1 of 25 ︰ 24）Mixed liquor, 12000rpm centrifugation 10min after 20min are rocked, are taken Clear liquid.Fen ︰ Lv Fang ︰ isoamyl alcohol mixed liquors are added, repeat aforesaid operations 3 times.Supernatant is taken again, adds what 2 times of volumes cooled down The sodium acetate of absolute ethyl alcohol and the 3mol/L of 1/10 volume, 12000rpm centrifugation 10min, abandoning supernatant, retains DNA precipitations, The precipitation is washed with 70% ethanol 2 times, after ethanol volatilization completely, with TE buffer solution DNA, and be diluted to 100ng/ μ again L, 4 DEG C of preservations.
2nd, the design of micro-satellite primers：On the basis of saladifish DNA sequence dna, the sequence of microsatellite DNA both sides is utilized In same species relative to the well-conserved of core sequence, accordingly in its design specific primers at both ends, this is amplified with it The microsatellite fragment in site.Because microsatellite core sequence mutation rate is of a relatively high, microsatellite DNA core sequence is caused to repeat secondary Several increases or decreases, i.e. the change of microsatellite DNA sequence length, and this is the root for detecting microsatellite polymorphism.The present invention's The specific primer sequence at microsatellite area core sequence both ends is：- the CCAAGAGCATCATATCGTGC -3 ' of normal chain 5 ', minus strand 5 '-TAGCCGCCACTTACACAGCC -3 ', the use of the annealing temperature during primer it is 53 DEG C.
3rd, PCR is expanded：It is loaded first, sample-adding amount is as follows：Saladifish 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；Primer (each 10pmol/ μ l), 1 μ l；Add aqua sterilisa to 25 μ l.Secondly, performing PCR reaction, its PCR amplification instrument program parameter are entered For：94 DEG C of denaturation 2min；94 DEG C of 30sec, 53 DEG C of 45sec, 72 DEG C of 40sec, 35 circulations；72 DEG C of extension 5min, 4 DEG C of preservations.
4th, the detection of PCR primer：PCR primer is separated by electrophoresis on 8% denaturing polyacrylamide gel, electrophoresis apparatus work( Rate is 15W, and electrophoresis time is 1.5 hours or so.After electrophoresis terminates, 30min is soaked with 10% glacial acetic acid solution first, then Distilled water flushing 5min, 0.1% silver nitrate solution immersion 30min, 3% sodium carbonate liquor colour developing 5min, finally with 10% Glacial acetic acid solution immersion 5min i.e. can obtain polymorphism collection of illustrative plates of the saladifish in Epinmoyn microsatellite core sequences, such as Shown in Fig. 1.As a result show, 20 individual coamplifications of saladifish go out 8 allele, illustrate microsatellite marker Epinmoyn has higher polymorphism, is appropriate for saladifish Genetic diversity evaluation, individual identification and genealogical identification Etc. research and application.
<110>University Of Yantai
<120>Utilize the microsatellite marker Epinmoyn primers detected to 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 detected using microsatellite marker Epinmoyn to saladifish genotype, it is characterised in that
Wherein described primer is sequence：
- the CCAAGAGCATCATATCGTGC-3 ' of forward primer 5 ',
- the TAGCCGCCACTTACACAGCC-3 ' of reverse primer 5 ', it is 53 DEG C with the annealing temperature during primer, wherein described micro- Satellite markers Epinmoyn core sequence is：gtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtat atatatatat atatatatat。
2. the method detected using microsatellite marker Epinmoyn to saladifish genotype, it is characterised in that first Extract the genomic DNA in saladifish blood and dilute standby；Recycle what is contained in saladifish DNA sequence dna micro- to defend Star core sequence, enter performing PCR amplification to the genomic DNA of saladifish Different Individual using specific primer, to PCR primer Denaturing polyacrylamide gel electrophoresis detection is carried out, so that it is determined that saladifish Different Individual is in Epinmoyn core sequences area Genotype, wherein described saladifish microsatellite marker Epinmoyn specific primer is：
- the CCAAGAGCATCATATCGTGC-3 ' of forward primer 5 ',
- the TAGCCGCCACTTACACAGCC-3 ' of reverse primer 5 ', it is 53 DEG C with the annealing temperature during primer, the microsatellite Mark Epinmoyn core sequence be：gtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtat atatatatat atatatatat。
3. detection method as claimed in claim 2, it is characterised in that enter to the genomic DNA of different saladifishs individual Capable PCR amplifications, its sample-adding parameter are：Each PCR reactions cumulative volume is 25 μ l, including 100ng saladifish genomes DNA；10 × PCR Buffer, 2.5 μ l；Mg2+1.5 mmol/L；Taq enzyme 1u；dNTP 0.1mmol/L；Specific primer each 10 pmol；Finally plus ddH2O to 25 μ l；Set PCR instrument program parameter be：94 DEG C of denaturation 2min；94 DEG C of 30sec, 53 DEG C 45 Sec, 72 DEG C of 40sec, the reaction carry out 35 circulations；72 DEG C of 5 min of extension, 4 DEG C of preservations.
4. detection method as claimed in claim 2, it is characterised in that denaturing polyacrylamide gel electricity is carried out to PCR primer The step of swimming detection：PCR primer is divided for 1.5 hours in 8% denaturing polyacrylamide gel with 15W invariable powers electrophoresis From, 30min is soaked with 10% glacial acetic acid solution, then distilled water flushing 5min, then with 0.1% silver nitrate solution immersion 30min, with 3% sodium carbonate liquor colour developing 5min, finally soak 5min with 10% glacial acetic acid solution.
5. application of the detection method between saladifish colony on genetic polymorphism map construction described in claim 2.
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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
3种石斑鱼微卫星标记的开发及跨种扩增;赵丽丽;《中国优秀硕士学位论文全文数据库》;20100531;第23页倒数第2段、第28-30页表3 *
Development of microsatellite markers for the kelp grouper Epinephelus bruneus by 454 pyrosequencing and transfer to related species;J.-H. Kang et al.;《Genetics and Molecular Research》;20131113;第12卷(第4期);5485-5493 *
石斑鱼遗传多样性的研究进展;尹绍武等;《水产科学》;20050831;第24卷(第8期);46-49 *

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