CN107419010A - Stalwart blood clam genotype detection primer and method based on microsatellite marker Scabrou_B - Google Patents
Stalwart blood clam genotype detection primer and method based on microsatellite marker Scabrou_B Download PDFInfo
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- CN107419010A CN107419010A CN201710503891.6A CN201710503891A CN107419010A CN 107419010 A CN107419010 A CN 107419010A CN 201710503891 A CN201710503891 A CN 201710503891A CN 107419010 A CN107419010 A CN 107419010A
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- blood clam
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q2600/156—Polymorphic or mutational markers
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Abstract
The invention discloses a kind of stalwart blood clam genotype detection primer and method based on microsatellite marker Scabrou_B.The genomic DNA in stalwart blood clam muscle is extracted first and is diluted standby；The microsatellite core sequence contained in stalwart blood clam DNA sequence dna is recycled to design specific primer；Then enter performing PCR using genomic DNA individual in primer pair chief blood clam difference proles or stalwart blood clam 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 stalwart blood clam.The present invention can efficiently obtain stalwart blood clam genetic marker Scabrou_B mapping genetic variations, and method is easy, and acquired results can intuitively detect the genotype of each individual of stalwart blood clam；Applied to structure of genetic marker, genealogical identification and genetic map etc. between individual in different proles or stalwart blood clam group.
Description
Technical field
The invention belongs to stalwart blood clam（Scapharca brouhtonii）DNA molecular Genetic Markers, it is related to a kind of detection
Stalwart blood clam microsatellite marker Scabrou_B technical method, specifically stalwart blood clam microsatellite marker Scabrou_B detection method.
Background technology
Stalwart blood clam（Scapharca brouhtonii）It is subordinate to Mollusca（Mollusca）, Bivalvia（Bivalvia）,
Blood clam mesh（Arcoida）, blood clam section（Arcidae）, blood clam category（Scapharca）, Ark Shell, blood shellfish, big hair clam are commonly called as, is a kind of large-scale
Economic shellfish is inhabited at bottom.Stalwart blood clam is distributed widely in west of pacific ocean bank, on the south Hokkaido, Japan, the Korea peninsula and Russia east
South.In China, stalwart blood clam is distributed mainly on the sea areas such as the Liaodong Peninsula southeast, Northern Shandong Peninsula and east.Stalwart blood clam belongs to big
Type blood clam, adult is substantially fertile, delicious meat, has very high economic value.Stalwart blood clam turns into the most important sea-farming in China
One of kind.However, at present there is larger dispute in the taxonomy of Northeast Asian region different geographic populations chief blood clam.Either exist
In formalness or on the sequence signature of mitochondrial genomes, different groups chief blood clam all show larger difference
(Yokogawa etc., 1997；Liu etc., 2013；Liu etc., 2014).The development of stalwart blood clam aquaculture is foster there is an urgent need to understand
Kind clearly genetic background and genetic diversity are grown, to grasp its resource situation, is laid the foundation for the development of stalwart blood clam aquaculture.
Microsatellite marker can solve the problem as a kind of codominant inheritance marks.Although isoenzyme mark, RAPD are marked and AFLP
Labelling technique can be used for studying the genetic structure and its genetic diversity of stalwart blood clam, be belonged to yet with these marks dominant
Genetic marker, detection efficiency are high not as codominant markers such as microsatellite markers.In addition, the shortcomings that isoenzyme mark is polymorphism
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, is characterized in that species is more, and allele number is more, and polymorphism is high, codominance,
Mendelian inheritance pattern, and be randomly distributed in genome, and microsatellite marker detection efficiency is high, as a result stablizes.But due to
The stalwart blood clam Polymorphism of Microsatellite Markers developed at present is not high, limits the development of its molecule genetics research, so there is an urgent need to
Obtain the microsatellite marker of high polymorphism with carry out the research of stalwart blood clam genetic diversity, individual identification and genealogical identification etc. and
Using.
Bibliography Yokogawa K. Morphological and genetic differences between
Japanese and Chinese red ark shell Scapharca broughtonii [J]. Fisheries
Science, 1997, 63(3): 332-337
Liu YG, Kurokawa T, Sekino M, et al. Complete mitochondrial DNA sequence
of the ark shell Scapharca broughtonii: an ultra-large metazoan mitochondrial
genome [J]. Comparative Biochemistry and Physiology part D, 2013, 8(1): 72-81
Liu YG, Kurokawa T, Sekino M, et al. Tandem repeat arrays in the
mitochondrial genome as a tool for detecting genetic differences among the
ark shell Scapharca broughtonii [J]. Marine Ecology-An Evolutionary
Perspective, 2014, 35(3): 273-280。
The content of the invention
It is an object of the invention to provide a kind of stalwart blood clam DNA molecular Genetic Markers of high polymorphism, i.e., stalwart blood clam microsatellite
Scabrou_B quick determination method is marked, to make up the deficiency of prior art.
The basic conception of the present invention is mainly using the microsatellite core sequence contained in stalwart blood clam DNA sequence dna, at its both ends
Design specific primer is gone forward side by side performing PCR detection, so as to rapidly detect hereditary variation of each individual of stalwart blood clam in this microsatellite area,
Obtain the primer（Microsatellite marker Scabrou_B）To the polymorphism collection of illustrative plates of stalwart blood clam, intuitively detected by collection of illustrative plates per each and every one
The genotype of body.Based on above-mentioned background present situation and actual requirement, the present invention obtains a microsatellite mark from stalwart blood clam DNA sequence dna
Note, is named as Scabrou_B, microsatellite marker Scabrou_B can be used for stalwart blood clam analysis of genetic diversity and genealogical identification.
The present invention is completed according to following operational aspect：The genomic DNA in stalwart blood clam muscle and dilute is extracted first
Release standby；The microsatellite core sequence contained in stalwart blood clam genomic dna sequence is recycled, design specificity is drawn at its sequence both ends
Thing；Then enter performing PCR using genomic DNA individual in primer pair chief blood clam difference proles or stalwart blood clam group to expand, PCR is produced
Thing carries out denaturing polyacrylamide gel electrophoresis detection, so that it is determined that the genotype of each individual, obtains the genetic polymorphism of stalwart blood clam
Property collection of illustrative plates.
Stalwart blood clam genomic DNA is extracted, 100ng/ μ l is diluted to, adds 1 μ l in each PCR reactions, react cumulative volume
For 25 μ l.
DNA sequence dna containing microsatellite sequence is：
ttataaacaa gtttgactgt atcataaaat atcacgaata ctgataattt
gtaagcgact tactttgttg tggcggtgat gtgttattgg aaactgaaat
CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT
CTCTCTCTCT CTcaacaagc gtgatatcag taaagtggtc ggtttgcctg
tttaatttgt gtatgggtga agtttcgaca tt
Wherein microsatellite core sequence is：CTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCT
CTCTCTCTCTCTCTCTCTCTCT
Scabrou_B micro-satellite primers sequences are：
- the ATATCACGAATACTGATAATTTG -3 ' of normal chain 5 ',
- the GAAACTTCACCCATACACAAATT -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 presented in stalwart blood clam Diversity Detection polymorphic
Property.
PCR amplification sample-adding parameter be：Each PCR reactions cumulative volume is 25 μ l, including 100ng chief's blood clam genomic DNAs；10
× PCR Buffer, 2.5 μ l；Mg2+1.5mmol/L；Tag enzymes 1u；Each 0.1mmol/L of dNTP；Each 10pmol of above-mentioned primer；
Add ddH2O to 25 μ l.Using during the primer set PCR instrument program parameter be：94 DEG C of denaturation 2min；94 DEG C of 30sec, 53 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 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 stalwart blood clam microsatellite marker Scabrou_B polymorphism collection of illustrative plates.
The beneficial effects of the invention are as follows the hereditary variation figure for the Scabrou_B microsatellite markers that can efficiently obtain stalwart blood clam
Spectrum, method are easy.Moreover, for other molecular genetic marker techniques, microsatellite marker meets Mendelian inheritance pattern,
In codominant inheritance, acquired results can intuitively detect the genotype of each individual of stalwart blood clam；And applied to different proles or
Structure of genetic marker, genealogical identification and genetic map etc. between individual in stalwart blood clam group.
Brief description of the drawings
Fig. 1 is that (numbering 1-20 is chief to the microsatellite marker Scabrou_B of the present invention detection collection of illustrative plates individual to stalwart blood clam 20
20 individuals of blood clam).
Embodiment
The present invention is described in detail below by embodiment in the heredity of stalwart blood clam Scabrou_B microsatellite core sequences DNA molecular
Labelling technique method.The genomic DNA in stalwart blood clam muscle is extracted first and is diluted standby；Recycle and contain in stalwart blood clam DNA sequence dna
Microsatellite core sequence, in its sequence design specific primers at both ends；Then using primer pair chief blood clam difference proles or
Individual genomic DNA enters performing PCR amplification in stalwart blood clam group, carries out denaturing polyacrylamide gel electrophoresis detection to PCR primer, really
The genotype of fixed each individual, that is, obtain the genetic polymorphism collection of illustrative plates of stalwart blood clam.
1st, the extraction of stalwart blood clam genomic DNA：100 μ l chief blood clams muscle are added in Eppendorf pipes, add cell cracking
Liquid 500 μ l and 20mg/ml the μ l of Proteinase K 5, gently shake up, and 37 DEG C overnight.Then 600 μ are added in cracked sample
L Fen Lv ︰ Fang ︰ isoamyl alcohol（The ︰ 1 of 25 ︰ 24）Mixed liquor, 12000rpm centrifugation 10min after 20min are rocked, take supernatant.Add
Fen ︰ Lv Fang ︰ isoamyl alcohol mixed liquors, repeat aforesaid operations 3 times.Supernatant is taken again, adds the absolute ethyl alcohol and 1/ of 2 times of volume coolings
The 3mol/L of 10 volumes sodium acetate, 12000rpm centrifugation 10min, abandoning supernatant, retains DNA precipitations, then with 70% ethanol
Wash the precipitation 2 times, after ethanol volatilization completely, with TE buffer solution DNA, and be diluted to 100ng/ μ l, 4 DEG C of preservations.
2nd, the design of micro-satellite primers：On the basis of stalwart blood clam DNA sequence dna, using the sequence of microsatellite DNA both sides same
Species, accordingly in its design specific primers at both ends, the site are amplified with it relative to the well-conserved of core sequence
Microsatellite fragment.Because microsatellite core sequence mutation rate is of a relatively high, the increasing of microsatellite DNA core sequence number of repetition is caused
Add deduct few, i.e. the change of microsatellite DNA sequence length, this is the root for detecting microsatellite polymorphism.The microsatellite of the present invention
The specific primer sequence at area core sequence both ends is：- the ATATCACGAATACTGATAATTTG -3 ' of normal chain 5 ', minus strand 5 ' -
GAAACTTCACCCATACACAAATT -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：Stalwart blood clam 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
Thing (each 10pmol/ μ l), 1 μ l；Add aqua sterilisa to 25 μ l.Secondly, performing PCR reaction is entered, its PCR amplification instrument program parameter is：94
DEG C 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 stalwart blood clam in Scabrou_B microsatellite core sequences, such as Fig. 1 institutes
Show.As a result as can be seen that 20 individual coamplifications of stalwart blood clam go out 18 allele, illustrate that microsatellite marker Scabrou_B has
There is very high polymorphism, be appropriate for the research of stalwart blood clam genetic diversity assessment, molecular ecology, individual identification etc. and answer
With.
<110>University Of Yantai
<120>Stalwart blood clam genotype detection primer and method based on microsatellite marker Scabrou_B
<160> 1
<210> 1
<211> 232
<212> DNA
<213>Stalwart blood clam（Scapharca brouhtonii）
<220>
<221> repeat_region
<222> (101)...(162)
<400> 1
ttataaacaa gtttgactgt atcataaaat atcacgaata ctgataattt gtaagcgact 1
tactttgttg tggcggtgat gtgttattgg aaactgaaat CTCTCTCTCT CTCTCTCTCT 61
CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CTcaacaagc gtgatatcag 121
taaagtggtc ggtttgcctg tttaatttgt gtatgggtga agtttcgaca tt 181
Claims (5)
1. a kind of stalwart blood clam microsatellite marker Scabrou_B genotype detection method, it is characterised in that extract stalwart blood clam muscle first
In genomic DNA and dilute standby；The microsatellite core sequence contained in stalwart blood clam DNA sequence dna is recycled, is drawn using specificity
Thing enters performing PCR amplification to the genomic DNA of stalwart blood clam Different Individual, and denaturing polyacrylamide gel electrophoresis inspection is carried out to PCR primer
Survey, so that it is determined that genotype of the stalwart blood clam Different Individual in Scabrou_B core sequences area.
2. one group of chief's blood clam microsatellite marker Scabrou_B detection primer, it is characterised in that the primer sequence is：
- the ATATCACGAATACTGATAATTTG -3 ' of normal chain 5 ',
- the GAAACTTCACCCATACACAAATT -3 ' of minus strand 5 ', the use of the annealing temperature during primer it is 53 DEG C.
3. stalwart blood clam microsatellite marker Scabrou_B as claimed in claim 1 detection method, it is characterised in that to different chiefs
The PCR amplifications that the genomic DNA of blood clam individual is carried out, its sample-adding parameter are：Each PCR reactions cumulative volume is 25 μ l, including 100ng
Stalwart blood clam genomic DNA；10 × PCR Buffer, 2.5 μ l；Mg2+1.5 mmol/L；Taq enzyme 1u；dNTP 0.1mmol/L；Right
It is required that each 10 pmol of specific primer described in 2；Finally plus ddH2O to 25 μ l；Set PCR instrument program parameter be：94℃
It is denatured 2min；94 DEG C of 30sec, 53 DEG C of 45sec, 72 DEG C of 40sec, the reaction carry out 35 circulations；72 DEG C extension 5 min, 4
DEG C preserve.
4. stalwart blood clam microsatellite marker Scabrou_B as claimed in claim 1 detection method, it is characterised in that to PCR primer
The step of carrying out denaturing polyacrylamide gel electrophoresis detection：By PCR primer in 8% denaturing polyacrylamide gel with
15W invariable powers electrophoresis is separated for 1.5 hours, soaks 30min with 10% glacial acetic acid solution, then distilled water flushing 5min,
Again with 0.1% silver nitrate solution soak 30min, with 3% sodium carbonate liquor develop the color 5min, finally with 10% glacial acetic acid
Solution soaks 5min.
5. application of the method between Kui Han colonies on genetic polymorphism map construction described in claim 1-4.
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| CN201710503891.6A CN107419010A (en) | 2017-06-28 | 2017-06-28 | Stalwart blood clam genotype detection primer and method based on microsatellite marker Scabrou_B |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561355A (en) * | 2015-02-01 | 2015-04-29 | 中国海洋大学 | Multiplex-PCR method for parentage assignment of scapharca broughtonii |
| CN105755165A (en) * | 2016-05-19 | 2016-07-13 | 烟台大学 | Primer and method for detecting paralichthys olivaceus microsatellite marked paraoliva-2 genotype |
-
2017
- 2017-06-28 CN CN201710503891.6A patent/CN107419010A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561355A (en) * | 2015-02-01 | 2015-04-29 | 中国海洋大学 | Multiplex-PCR method for parentage assignment of scapharca broughtonii |
| CN105755165A (en) * | 2016-05-19 | 2016-07-13 | 烟台大学 | Primer and method for detecting paralichthys olivaceus microsatellite marked paraoliva-2 genotype |
Non-Patent Citations (3)
| Title |
|---|
| 孙楠等: "利用微卫星标记的魁蚶混交家系鉴定", 《中国海洋大学学报》 * |
| 田吉腾: "魁蚶微卫星富集文库的构建及四个地理群体的遗传多样性分析", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 高悦勉等: "魁蚶遗传多样性的微卫星分析", 《2010年中国水产学会学术年会论文摘要集》 * |
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