CN104975012B - Great flounder microsatellite molecular marker site and primer - Google Patents
Great flounder microsatellite molecular marker site and primer Download PDFInfo
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- CN104975012B CN104975012B CN201510380762.3A CN201510380762A CN104975012B CN 104975012 B CN104975012 B CN 104975012B CN 201510380762 A CN201510380762 A CN 201510380762A CN 104975012 B CN104975012 B CN 104975012B
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Abstract
The present invention provides 11 microsatellite molecular marker sites of great flounder and its polymorphism primers, the amplification of obtained primer has the polymorphism and stability of height, the identification of affiliation between the genetic diversity and population of great flounder provides data, makes up the deficiencies in the prior art.
Description
Technical field
The invention belongs to molecular biology DNA molecular marker technical fields, and in particular to great flounder micro-satellite molecule mark
The exploitation of note.
Background technique
Great flounder (Platichthys stellatus) is also known as asropyle platichthys stellatus pallas, is subordinate to Pleuronectiformes Pangasiidae flounder category, is wide
Salt, cold aqueous type are distributed in China, Korea, South Korea, Japan, Russia and the U.S., sea area, pacific rim, in China Jilin
The TUMENJIANG RIVER of province is also distributed.Since great flounder nutritive value is high, unique in taste, delicious flavour, so market value is high,
Domestic and international market is well received.Currently, the research report in relation to great flounder is also fewer both at home and abroad, it is concentrated mainly on biology
Status, ecologicaI distribution and quantity qf population resources.In addition, Japan and French scholar utilize Isozyme Analysis wild population star spot river
The Genetic Constitution of Population of flounder studies its physiology, biochemistry etc. there are also some scholars, but at present for great flounder
Research in terms of microsatellite is then few, and the SSR sequence for the great flounder announced on NCBI only has 158, this is to a certain degree
On limit the genetic breeding and quality-improving of great flounder.
Microsatellite marker (SSRs:simple sequence repeats) or Short tandem repeatSTR (Short tandem
Repeats, STRS), it is a kind of DNA molecular marker of widely applied genetic arts.SSRs is to be widely present eucaryote
Simple repetition DNA segment in genome, general each recurring unit only 1-6 base, repeat number is 10-20 times, wherein moving
It is most commonly seen with dinucleotide (CA/GT) n in object.Microsatellite has polymorphism high, and the hereditary information provided is more, PCR amplification
Effect is reproducible, and in genome the advantages that dispersed distribution, is usually used in the analysis of genetic diversity of biology, genetic map
With crossbreeding etc..Low to sample requirement since the quantity of DNA used in microsatellite research is few, therefore, microsatellite is analyzed in heredity
Field is with a wide range of applications.
Summary of the invention
The object of the present invention is to provide 11 microsatellite molecular marker sites of great flounder and its polymorphism primers, are star
The identification of affiliation between the genetic diversity and population of spot flounder provides data, makes up the deficiencies in the prior art.
The present invention filters out 11 microsatellite locus using high throughput sequencing technologies from great flounder DNA genome,
Nucleotide sequence is PTS:1-11 respectively, and sequence is SEQ ID:1-11.
Microsatellite sequence further includes under strict conditions with above-mentioned nucleotide sequence hybridization, includes identical microsatellite weight
Multiple nucleotide sequence molecule.
Micro-satellite primers of the present invention are designed just from the flanking sequence at the microsatellite both ends of sequence SEQ ID:1-11
To, reverse primer.Micro-satellite primers sequence is SEQ ID:12-33 respectively
Microsatellite locus, site sequence and corresponding primer information such as table 1:
Table 1
| Site | Microsatellite sequence | Forward primer sequence | Reverse primer void column |
| PTS1 | PTS ID NO:1 | PTS ID NO:12 | PTS ID NO:13 |
| PTS2 | PTS ID NO:2 | PTS ID NO:14 | PTS ID NO:15 |
| PTS3 | PTS ID NO:3 | PTS ID NO:16 | PTS ID NO:17 |
| PTS4 | PTS ID NO:4 | PTS ID NO:18 | PTS ID NO:19 |
| PTS5 | PTS ID NO:5 | PTS ID NO:20 | PTS ID NO:21 |
| PTS6 | PTS ID NO:6 | PTS ID NO:22 | PTS ID NO:23 |
| PTS7 | PTS ID NO:7 | PTS ID NO:24 | PTS ID NO:25 |
| PTS8 | PTS ID NO:8 | PTS ID NO:26 | PTS ID NO:17 |
| PTS9 | PTS ID NO:9 | PTS ID NO:28 | PTS ID NO:19 |
| PTS10 | PTS ID NO:10 | PTS ID NO:30 | PTS ID NO:31 |
| PTS11 | PTS ID NO:11 | PTS ID NO:32 | PTS ID NO:33 |
Microsatellite polymorphism primer of the invention is used for great flounder Diversity Detection, comprising the following steps:
1) extraction of genomic DNA: Tiangeng marine animal tissue DNA extracts kit is used
2) microsatellite PCR expands: expanding great flounder gene using the micro-satellite primers of FAM, HEM, TAMRA fluorescent marker
Group DNA, obtains its amplified production.
3) amplified production electrophoresis: the electrophoresis on Capillary Electrophoresis Genetic Analyser makees molecular weight internal standard with GS500LIZ, a
Body amplified production is with peak Scanner Software (v1.0) to Capillary Electrophoresis data preliminary treatment.
4) analysis of genetic diversity: taking according to the size of the molecular weight of each individual microsatellite amplified production and determine genotype,
Genetic diversity parameter is calculated using PopGene 32.
The present invention filters out 11 microsatellite locus from great flounder genomic DNA, and according to these sites in Wei Wei
The flanking sequence at championship point both ends designs specific primer, has polymorphism and stabilization using the amplification of obtained primer
Property, it is applicable to the detection of great flounder population genetic diversity, Individual identification and marker assisted selection field.
Specific embodiment
The screening of embodiment one, microsatellite molecular marker
The ssr analysis that the present invention carries out in the transcript profile library according to the great flounder brain tissue of building compares transcript profile
Unigene in library screens to have obtained the site of 50 microsatellite molecular markers, and according to related locus, devises 50 pairs
Screening primer, verified in 30 individuals of great flounder.
The verifying of embodiment two, micro-satellite primers
Using great flounder genome as template, temperature gradient PCR is carried out with 50 pairs of primers to find out optimum annealing temperature,
1% agarose gel electrophoresis detects PCR amplification result.12 pairs are picked out in the primer for having found out optimum annealing temperature again
Synthesize the fluorescent dye primer of FAM, HEM, TAMRA label.20 μ L system PCR:ddH2O, 13 μ L；HF Buffer 4μL；
dNTPs(2.5mM)1.6μL；0.5 μ L of upstream primer (10mM)；0.5 μ L of downstream primer (10mM)；0.28 μ L of DNA profiling；
Thermo Scientific Phusion DNA polymerase(2U/μL)0.12μL.PCR program: 98 DEG C of 30s, (98 DEG C
10s, Tm 30s, 72 DEG C of 20s) (the upper Shanghai's style is gloomy with the full-automatic DNA sequencer of ABI3730xl by x30 cycle, 72 DEG C of 10min.
Promise Biotechnology Co., Ltd) sequencing.Capillary Electrophoresis data are tentatively located with Peak Scanner Software (v1.0)
Reason.Software PopGene 32 carries out Heredity index (allele (na:Observed number of alleles), effective etc.
Position gene (ne:Effective number of alleles), observation heterozygosity (Observed heterozygosity), phase
Hope heterozygosity (Expected heterozygosity), Shannon heritability index (Shannon ' s Information index
(I)), NeiShi diversity indices (Nei ' s gene diversity index (Nei)), polymorphism information content
(polymorphism information content (PIC))) it calculates.
The application of embodiment three, great flounder micro-satellite primers in close species genetic diversity
1. firstly, the marine animal tissue gene group DNA extraction kit using Tiangeng (TIANGEN) biotech firm is extracted
The genomic DNA of great flounder.Utilize the concentration and quality of spectrophotometry and 1% agarose gel electrophoresis detection DNA.
2. being expanded with the primer PCR of FAM, HEM, TAMRA fluorescent marker, 20 μ L system PCR:ddH2O, 13 μ L；HF
Buffer 4μL；dNTPs(2.5mM)1.6μL；0.5 μ L of upstream primer (10mM)；0.5 μ L of downstream primer (10mM)；DNA profiling
0.28μL；Thermo Scientific Phusion DNA polymerase(2U/μL)0.12μL.PCR program: 98 DEG C
30s, (98 DEG C of 10s, Tm 30s, 72 DEG C of 20s) x30 cycle, 72 DEG C of 10min. Capillary Electrophoresis (upper Shanghai's style Sen Nuosheng
Object Science and Technology Ltd.) detection.
3. with Peak Scanner Software (v1.0) to Capillary Electrophoresis data preliminary treatment.Software PopGene
32 carry out Heredity index measurement.(allele (na:Observed number of alleles), effective number of alleles (ne:
Effective number of alleles), observation heterozygosity (Observed heterozygosity), expectation heterozygosity
(Expected heterozygosity), Shannon heritability index (Shannon ' s Information index (I)), NeiShi
Diversity indices (Nei ' s gene diversity index (Nei)), polymorphism information content (polymorphism
Information content (PIC))) it calculates.
The recurring unit of 2:11 microsatellite locus of table, primer relevant information
Wherein Tm is the annealing temperature of microsatellite, and na is number of alleles, and ne is effective number of allele, and Obs_Het is seen
Hope that heterozygosity, Exp-Het are desired heterozygosity.PIC is polymorphism information content, and I is shannon index, and Nei is that NeiShi diversity refers to
Number, PIC is polymorphism information content.
11 pairs of primer pairs, 30 great flounder genomic DNAs of the invention expand, analysis of genetic diversity result table
The allele number of bright each microsatellite locus is differed from 3 to 6, and average number of alleles is 4.4545, average effective equipotential
Gene number is 2.4384, averagely looks around heterozygosity 0.5273, and average expectation heterozygosity is 0.5736.Micro-satellite primers of the invention
It can be used for great flounder Diversity Detection, Inheritance of Quantitative Characters map, genetic linkage left figure and family and individual mirror
It is fixed.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (7)
1. great flounder microsatellite molecular marker, it is characterised in that sequence table SEQ .ID.No.1 is any into SEQ.ID.No.11
Base sequence, they distinguish marker numbers be PT1, PT2, PT3, PT4, PT5, PT6, PT7, PT8, PT9, PT10, PT11.
2. the combination of great flounder microsatellite molecular marker described in claim 1, it is characterised in that be selected from great flounder microsatellite
Two or more of molecular labeling PT1, PT2, PT3, PT4, PT5, PT6, PT7, PT8, PT9, PT10, PT11.
3. the great flounder micro-satellite primers designed on the flanking sequence at microsatellite both ends described in claim 1, feature
Be: the micro-satellite primers sequence is SEQ ID NO:12-33, and wherein sequence 12 and 13 is for expanding PT1 sequence, sequence
14 and 15 for expanding PT2 sequence, and so on.
4. the combination of great flounder micro-satellite primers described in claim 3, it is characterised in that selected from amplification PT1 to PT11 primer pair
One group or two groups.
5. great flounder microsatellite molecular marker of any of claims 1 or 2 or combinations thereof is more in the population genetic of great flounder
Application in terms of sample detection, Individual identification or marker assisted selection.
6. primer of the great flounder microsatellite molecular marker of claim 3 or 4 or combinations thereof is lost in the population of great flounder
Pass the application in terms of diversity detection, Individual identification or marker assisted selection.
7. application according to claim 5 or 6, it is characterised in that the Diversity Detection of great flounder includes following step
It is rapid:
1) extraction of genomic DNA: Tiangeng marine animal tissue DNA extracts kit is used；
2) microsatellite PCR expands: expanding great flounder genome using the micro-satellite primers of FAM, HEM, TAMRA fluorescent marker
DNA obtains its amplified production；
3) amplified production electrophoresis: the electrophoresis on Capillary Electrophoresis Genetic Analyser makees molecular weight internal standard with GS500LIZ, and individual expands
Increase production object with peak Scanner Software (v1.0) to Capillary Electrophoresis data preliminary treatment；
4) it analysis of genetic diversity: is taken according to the size of the molecular weight of each individual microsatellite amplified production and determines genotype, utilized
PopGene 32 calculates genetic diversity parameter.
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| CN108103229B (en) * | 2018-01-22 | 2021-09-07 | 中国医学科学院北京协和医院 | Candida krusei STR molecular marker and application thereof |
| CN110257533B (en) * | 2019-08-01 | 2020-10-27 | 南京林业大学 | Microsatellite marker locus of bullfight and primer thereof |
| CN110760599B (en) * | 2019-12-16 | 2020-10-02 | 吉林省水产科学研究院 | Cannabis harfish microsatellite molecular marker locus, polymorphism primer and application |
| CN112831570B (en) * | 2021-02-08 | 2022-02-01 | 南京林业大学 | Black iso-striated lipocarp microsatellite marker locus and general application thereof in fishes of the genus and the near-source genus |
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| CN101270389A (en) * | 2008-05-09 | 2008-09-24 | 中国水产科学研究院黄海水产研究所 | Cynoglossus semilaevis special numerator mark and uses thereof |
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| CN103305508A (en) * | 2013-06-03 | 2013-09-18 | 中国水产科学研究院黄海水产研究所 | Marbled flounder microsatellite locus and primer |
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| 《Genetic diversity and differentiation of the Korean starry flounder (Platichthys stellatus) between and within cultured stocks and wild populations inferred from microsatellite DNA analysis》;Hye Suck An等;《Molecular Biology reports》;20141231;第14卷(第11期);全文 |
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