CN105779614B - Utilize the Xinjiang Alaska grayling hereditary variation detection method of microsatellite marker Thymalag - Google Patents
Utilize the Xinjiang Alaska grayling hereditary variation detection method of microsatellite marker Thymalag Download PDFInfo
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- CN105779614B CN105779614B CN201610241405.3A CN201610241405A CN105779614B CN 105779614 B CN105779614 B CN 105779614B CN 201610241405 A CN201610241405 A CN 201610241405A CN 105779614 B CN105779614 B CN 105779614B
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Abstract
The invention discloses a kind of Xinjiang Alaska grayling hereditary variation detection methods using microsatellite marker Thymalag.The genomic DNA in Xinjiang Alaska grayling muscle is extracted first and is diluted spare；The microsatellite core sequence contained in Xinjiang Alaska grayling DNA sequence dna is recycled, in its sequence design specific primers at both ends；Then PCR amplification is carried out using genomic DNA individual in the primer pair Xinjiang Alaska grayling difference proles or Xinjiang Alaska grayling group, PCR product is detected；It is analyzed using the band that product occurs, determines the genotype of each individual, and obtain the genetic polymorphism map of Xinjiang Alaska grayling.It is characteristic of the invention that the mapping genetic variations of Xinjiang Alaska grayling genetic marker Thymalag can be obtained efficiently, method is easy.
Description
Technical field
The invention belongs to Xinjiang Alaska grayling (Thymallus arcticus grubei) DNA molecular Genetic Markers,
It is related to a kind of technical method for detecting Xinjiang Alaska grayling microsatellite marker Thymalag, specifically Xinjiang Alaska grayling microsatellite
Mark the hereditary variation detection method of Thymalag.
Background technique
Xinjiang Alaska grayling (Thymallus arcticus grubei) belong to salmon shape mesh (Salmoniformes), salmonidae
(Salmonidae), grayling subfamily (Thymallinae), grayling category (Thymallus), it is commonly called as careless spot, oil mark also known as stick flower
Fish, flower shark's fin.Body is elongated, flat-sided, and dorsal fin is long and tall and big, upper limb boss is in flag shape the line of several russet spot formations
Band, body colour are bright-coloured.It is each that wild resource is mainly distributed on Altay, Xinjiang city Fuyun County and the upstream in Burqin County Eerqisihe River
In tributary.Alaska grayling belongs to the high oxygen consumption fish of typical cold water, lives in mountain stream streams throughout the year, and water quality is limpid pollution-free, and The turbulent river crashes its way through,
The pebbly place in river bed.Alaska grayling appearance is magnificent, and meat is especially fine and smooth, is not only rare Xinjiang Endemic fish, and
The important Fish Germplasm Resources in China, there is high economic development value.Due to environmental pollution, forest deterioration and mistake in recent years
It crosses human factors, the resources of the fish such as fishing and faces exhaustion, II class of Xinjiang is classified as by the People's Government, Xinjiang Uygur Autonomous Regions
Important aquatic wild protection animal.In recent years, Production and Construction Corps of Xinjiang's Fishery technical Center for Popularization to big equal researchers
Had begun Alaska grayling artificial breeding, resource increment and artificial domesticating and cultivating research, and achieve certain research at
Fruit is expected to realize the industrialization of cultivation.For Xinjiang Alaska grayling as a kind of emerging breed variety, genetics research is relatively thin
Weak, there are no the reports of microsatellite marker exploitation.Although isoenzyme mark, RAPD label and AFLP labelling technique can be used for
The genetic structure and its genetic diversity of Xinjiang Alaska grayling are studied, however since these labels belong to dominant inheritance label,
Detection efficiency is high not as good as codominant markers such as microsatellite markers.In addition, the shortcomings that isoenzyme mark is that polymorphism is low, RAPD mark
The stability of note is bad, and AFLP marking operation is cumbersome, these all greatly limit their application.And microsatellite sequence divides extensively
It is distributed in eukaryotic gene group, its main feature is that type is more, allele number is more, and polymorphism is high, codominance, Mendelian inheritance
Mode, and be randomly distributed in genome, and microsatellite marker detection efficiency is high, it is as a result stable.But due to lacking Xin Jiangbei
Pole grayling microsatellite marker, limits the development of its molecule genetics research, thus there is an urgent need to obtain microsatellite marker with into
The research and application of row Xinjiang Alaska grayling genetic diversity, individual identification and genealogical identification etc..
Summary of the invention
The object of the present invention is to provide a kind of Xinjiang Alaska grayling DNA molecular Genetic Markers of high polymorphism, i.e., newly
The rapid detection method of boundary Alaska grayling microsatellite marker Thymalag, to make up the deficiency of prior art.
Basic conception of the invention mainly utilizes the microsatellite core sequence contained in the Alaska grayling DNA sequence dna of Xinjiang,
In its design specific primers at both ends and PCR detection is carried out, so that rapidly the detection each individual of Xinjiang Alaska grayling is micro- herein
The hereditary variation in satellite area obtains the primer (microsatellite marker Thymalag) to the polymorphism map of Xinjiang Alaska grayling, leads to
Cross the genotype that map intuitively detects each individual.Status and actual requirement based on the above background, the present invention is from Xin Jiangbei
A microsatellite marker is obtained in the grayling DNA sequence dna of pole, is named as Thymalag, and microsatellite marker Thymalag can be used for
Xinjiang Alaska grayling analysis of genetic diversity and genealogical identification.
The present invention is completed according to following operational aspect: the genome first in extraction Xinjiang Alaska grayling muscle
DNA simultaneously dilutes spare；The microsatellite core sequence contained in Xinjiang Alaska grayling genomic dna sequence is recycled, in its sequence
Design specific primers at both ends；Then using in the primer pair Xinjiang Alaska grayling difference proles or Xinjiang Alaska grayling group
The genomic DNA of body carries out PCR amplification, denaturing polyacrylamide gel electrophoresis detection is carried out to PCR product, so that it is determined that each
The genotype of individual obtains the genetic polymorphism map of Xinjiang Alaska grayling.
Xinjiang Alaska grayling genomic DNA is extracted, 100ng/ μ l is diluted to, 1 μ l is added in each PCR reaction, instead
Answering total volume is 25 μ l.
DNA sequence dna containing microsatellite sequence are as follows:
1 cccctgttac ttgttggcgt aacaaagtgt gtgtgtttgt gtgtgtgtgt gtttgtgtgt
61 gtttttctgt gtgcagggaa acacacttgt tccttgacac tgctttgaag acactttatg
121 ttggggtttc ctatattttg gcagttacct gtagctatct gtaggaaatt cctcacactt
181 gcccattatc actttttaaa ttcaacctcc tcttagtatt ttaatggaca caccatcatg
241 atagattcat ctctaacgag caatagttaa cagaaatacc cttcctttat tcaagccaaa
301 ctctaaacag tttaaactat ctatccagct tcactgcatc tctgtttcca c
Wherein microsatellite core sequence are as follows: TGTGTGTGTGTGTG
Thymalag micro-satellite primers sequence are as follows:
Normal chain 5 '-CCCTGTTACTTGTTGGCGTAA -3 ',
Minus strand 5 '-TGGGCAAGTGTGAGGAATTT -3 ', annealing temperature when using the primer are 58 DEG C.
Microsatellite core sequence and primer sequence are core of the invention, in the Alaska grayling Diversity Detection of Xinjiang
Polymorphism is presented.
The sample-adding parameter of PCR amplification are as follows: each PCR reaction total volume is 25 μ l, including the Xinjiang 100ng Alaska grayling gene
Group DNA；10 × PCR Buffer, 2.5 μ l；Mg2+1.5mmol/L；Tag enzyme 1u；Each 0.1mmol/L of dNTP；Above-mentioned primer is each
10pmol；Add ddH2O to 25 μ l.Use the program parameter that PCR instrument is set when the primer are as follows: 94 DEG C of denaturation 2min；94℃
30sec, 58 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 product: by PCR product with 15W invariable power electrophoresis in 8% denaturing polyacrylamide gel
It is separated within 1.5 hours.After electrophoresis, 30min is impregnated with 10% glacial acetic acid solution first, then distilled water flushing
5min, then with 0.1% silver nitrate solution impregnate 30min, with 3% sodium carbonate liquor develop the color 5min, finally with 10% ice
Acetum impregnates 5min, and the polymorphism map of Xinjiang Alaska grayling microsatellite marker Thymalag can be obtained.
Therefore, it is characteristic of the invention that the something lost of the Thymalag microsatellite marker of Xinjiang Alaska grayling can be obtained efficiently
The different map of the progress of disease, method are easy.Moreover, microsatellite marker meets Mendel for other molecular genetic marker techniques
Hereditary pattern, is in codominant inheritance, and acquired results can intuitively detect the genotype of each individual of Xinjiang Alaska grayling；And it answers
For genetic marker, genealogical identification and building of genetic map etc. between individual in different proles or Xinjiang Alaska grayling group.
Detailed description of the invention
Fig. 1 is microsatellite marker Thymalag of the present invention to Xinjiang Alaska grayling 20 individual test map (numbers
1-20 be 20 individuals of Xinjiang Alaska grayling).
Specific embodiment
The present invention is described in detail below by embodiment at Xinjiang Alaska grayling Thymalag microsatellite core sequence DNA points
Sub- Genetic Markers method.The genomic DNA in Xinjiang Alaska grayling muscle is extracted first and is diluted spare；Recycle Xinjiang
The microsatellite core sequence contained in Alaska grayling DNA sequence dna, in its sequence design specific primers at both ends；Then drawn using this
Object carries out PCR amplification to genomic DNA individual in Xinjiang Alaska grayling difference proles or Xinjiang Alaska grayling group, to PCR
Product carries out denaturing polyacrylamide gel electrophoresis detection, determines the genotype of each individual, that is, obtains Xinjiang Alaska grayling
Genetic polymorphism map.
1, Eppendorf pipe the extraction of Xinjiang Alaska grayling genomic DNA: is added in 100 Xinjiang μ g Alaska grayling muscle
In, the 5 μ l of Proteinase K of cell pyrolysis liquid 500 μ l and 20mg/ml are added, is gently shaken up, 37 DEG C are overnight.Then it is cracking
600 μ l Fen Lv ︰ Fang ︰ isoamyl alcohol (25 ︰, 24 ︰ 1) mixed liquors are added in good sample, 12000rpm is centrifuged after shaking 20min
10min takes supernatant.Fen ︰ Lv Fang ︰ isoamyl alcohol mixed liquor is added, is repeated aforesaid operations 3 times.Supernatant is taken again, is added 2 times
The sodium acetate of the cooling dehydrated alcohol of volume and the 3mol/L of 1/10 volume, 12000rpm are centrifuged 10min, discard supernatant liquid, protect
DNA is stayed to precipitate, then with 70% ethanol washing the precipitating 2 times, after ethyl alcohol volatilization completely, with TE buffer solution DNA, and it is dilute
It is interpreted as 100ng/ μ l, 4 DEG C of preservations.
2, the design of micro-satellite primers: in Xinjiang on the basis of Alaska grayling DNA sequence dna, the sequence of microsatellite DNA two sides is utilized
Same species are listed in relative to the well-conserved of core sequence, accordingly in its design specific primers at both ends, are amplified with it
The microsatellite segment in the site.Since microsatellite core sequence mutation rate is relatively high, microsatellite DNA core sequence is caused to repeat
Number increases or decreases, i.e. the variation of microsatellite DNA sequence length, this is the root for detecting microsatellite polymorphism.The present invention
Microsatellite area core sequence both ends specific primer sequence are as follows: normal chain 5 '-CCCTGTTACTTGTTGGCGTAA-3 ', bear
Chain 5 '-TGGGCAAGTGTGAGGAATTT-3 ', annealing temperature when using the primer are 58 DEG C.
3, PCR amplification: being loaded first, and sample-adding amount is as follows: Xinjiang Alaska grayling 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, carrying out PCR reaction, PCR amplification instrument program parameter
Are as follows: 94 DEG C of denaturation 2min；94 DEG C of 30sec, 58 DEG C of 45sec, 72 DEG C of 40sec, 35 circulations；72 DEG C of extension 5min, 4 DEG C of preservations.
4, the detection of PCR product: PCR product is separated by electrophoresis on 8% denaturing polyacrylamide gel, electrophoresis apparatus function
Rate is 15W, and electrophoresis time is 1.5 hours or so.After electrophoresis, 30min is impregnated with 10% glacial acetic acid solution first, then
Distilled water flushing 5min, 0.1% silver nitrate solution impregnate 30min, 3% sodium carbonate liquor colour developing 5min, finally with 10%
Glacial acetic acid solution impregnate 5min and Xinjiang Alaska grayling can be obtained in the polymorphism map of Thymalag microsatellite core sequence,
As shown in Figure 1.Fig. 1 the result shows that, 20 of Xinjiang Alaska grayling individual coamplifications go out 8 allele, illustrate microsatellite mark
Remember Thymalag polymorphism with higher, is appropriate for Xinjiang Alaska grayling genetic diversity and genetic structure analysis, individual
The research and application of identification and genealogical identification etc..
<110>Xinjiang University
<120>the Xinjiang Alaska grayling hereditary variation detection method of microsatellite marker Thymalag is utilized
<160> 1
<210> 1
<211> 351
<212> DNA
<213>Xinjiang Alaska grayling (Thymallus arcticus grubei)
<220>
<221> repeat_region
<222> (38)...(51)
<400> 1
1 cccctgttac ttgttggcgt aacaaagtgt gtgtgtttgt gtgtgtgtgt gtttgtgtgt
61 gtttttctgt gtgcagggaa acacacttgt tccttgacac tgctttgaag acactttatg
121 ttggggtttc ctatattttg gcagttacct gtagctatct gtaggaaatt cctcacactt
181 gcccattatc actttttaaa ttcaacctcc tcttagtatt ttaatggaca caccatcatg
241 atagattcat ctctaacgag caatagttaa cagaaatacc cttcctttat tcaagccaaa
301 ctctaaacag tttaaactat ctatccagct tcactgcatc tctgtttcca c
Claims (5)
1. one group of primer detected using microsatellite marker Thymalag to Xinjiang Alaska grayling hereditary variation, feature are existed
In,
Wherein the primer is sequence:
Normal chain 5 '-CCCTGTTACTTGTTGGCGTAA-3 ',
Minus strand 5 '-TGGGCAAGTGTGAGGAATTT-3 ', annealing temperature when using the label are 58 DEG C.
2. the method detected using microsatellite marker Thymalag to Xinjiang Alaska grayling hereditary variation, which is characterized in that
The genomic DNA in Xinjiang Alaska grayling muscle is extracted first and is diluted spare；It recycles and contains in Xinjiang Alaska grayling DNA sequence dna
Some microsatellite core sequences carry out PCR expansion using genomic DNA of the specific primer to Xinjiang Alaska grayling Different Individual
Increase, denaturing polyacrylamide gel electrophoresis detection is carried out to PCR product, so that it is determined that Xinjiang Alaska grayling Different Individual exists
The genotype in Thymalag core sequence area, wherein the specificity of the Xinjiang Alaska grayling microsatellite marker Thymalag is drawn
Object are as follows:
Normal chain 5 '-CCCTGTTACTTGTTGGCGTAA-3 ',
Minus strand 5 '-TGGGCAAGTGTGAGGAATTT-3 ', annealing temperature when using the label are 58 DEG C.
3. being detected as claimed in claim 2 using microsatellite marker Thymalag to Xinjiang Alaska grayling hereditary variation
Method, which is characterized in that the PCR amplification that the genomic DNA of different Xinjiang Alaska grayling individuals carries out, be loaded parameter are as follows:
Each PCR reaction total volume is 25 μ L, including the Xinjiang 100ng Alaska grayling genomic DNA；10 × PCR Buffer, 2.5 μ L；
Mg2+1.5 mmol/L；Taq enzyme 1u；dNTP 0.1mmol/L；Each 10 pmol of specific primer in claim 2；Finally plus
ddH2O to 25 μ L；The program parameter of PCR instrument is set are as follows: 94 DEG C of denaturation 5min；94 DEG C of 45sec, 58 DEG C of 1min, 72 DEG C
40sec, the reaction carry out 35 circulations；72 DEG C of 5 min of extension, 4 DEG C of preservations.
4. being detected as claimed in claim 2 using microsatellite marker Thymalag to Xinjiang Alaska grayling hereditary variation
Method, which is characterized in that the step of denaturing polyacrylamide gel electrophoresis detection is carried out to PCR product: by PCR product 8%
Denaturing polyacrylamide gel in separated within 1.5 hours with 15W invariable power electrophoresis, with 10% glacial acetic acid solution impregnate
30min, then distilled water flushing 5min, then 30min is impregnated with 0.1% silver nitrate solution, it is developed the color with 3% sodium carbonate liquor
5min finally impregnates 5min with 10% glacial acetic acid solution.
5. method described in any one of claim 2-4 is between the Alaska grayling group of Xinjiang on genetic polymorphism map construction
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| 茴鱼微卫星标记开发及保护遗传学研究进展;刘云国等;《水产科学》;20160131;第35卷(第1期);全文 |
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