CN105779614A - Sinkiang Alaska grayling heritable variation detection method utilizing microsatellite marker Thymalag - Google Patents
Sinkiang Alaska grayling heritable variation detection method utilizing microsatellite marker Thymalag Download PDFInfo
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Abstract
The invention discloses a Sinkiang Alaska grayling heritable variation detection method utilizing a microsatellite marker Thymalag. The method comprises the steps that firstly, a genome DNA in Sinkiang Alaska grayling muscle is extracted and diluted for use; then, a microsatellite core sequence contained in a Sinkiang Alaska grayling DNA sequence is utilized, and specific primers are designed at the two ends of the sequence; then, the primers are used for performing PCR amplification on genome DNA of different geographic stocks or individuals inside Sinkiang Alaska grayling, and a PCR product is detected; strips of the product are analyzed, the gene type of each individual is determined, and a genetic polymorphic map of Sinkiang Alaska grayling is obtained. The method has the advantages that the genetic variation map of the genetic marker Thymalag of Sinkiang Alaska grayling can be obtained rapidly, and the method is simple and convenient to implement.
Description
Technical field
The invention belongs to Xinjiang Alaska grayling (Thymallusarcticusgrubei) DNA molecular Genetic Markers, relate to a kind of technical method detecting Xinjiang Alaska grayling microsatellite marker Thymalag, specifically the hereditary variation detection method of Xinjiang Alaska grayling microsatellite marker Thymalag.
Background technology
Xinjiang Alaska grayling (Thymallusarcticusgrubei) belongs to salmon shape order (Salmoniformes), salmon section (Salmonidae), grayling subfamily (Thymallinae), grayling genus (Thymallus), it is commonly called as grass speckle, oil mark, has another name called rod Percocypris pingi (Tchang), flower shark's fin.Body is elongated, flat-sided, dorsal fin length and tall and big, and upper limb boss, in flag shape, has the stricture of vagina band of several russet spot formations, and body colour is bright-coloured.Wild resource is mainly distributed in each tributary, upstream in Fuyun County, Altay, Xinjiang city and Eerqisihe River, Burqin County.Alaska grayling belongs to typical case cold water height oxygen consumption fish, lives in mountain stream streams throughout the year, and water quality is limpid pollution-free, and The turbulent river crashes its way through, pebbly place, river bed.Alaska grayling outward appearance is magnificent, and meat is especially fine and smooth, is not only famous and precious Xinjiang Endemic fish, the Fish Germplasm Resources that Ye Shi China is important, has high economic development value.Owing to environmental pollution in recent years, forest deterioration and transition such as fish at the anthropic factor, the resource of this fish faces exhaustion, is classified as the important aquatic wild protection animal of Xinjiang II class by the People's Government of Xinjiang Uygur Autonomous Regions.In recent years, the research worker such as the Xiang Wei of Production and Construction Corps of Xinjiang's Fishery technical Center for Popularization has had begun to the artificial breeding of Alaska grayling, resource increment and artificial domesticating and cultivating research, and achieves certain achievement in research, is expected to realize the industrialization of cultivation.Xinjiang Alaska grayling is as a kind of emerging breed variety, and genetics research is relatively weak, but without the report of microsatellite marker exploitation.Although isoenzyme mark, RAPD labelling and AFLP labelling technique can be used for hereditary constitution and the genetic diversity thereof of research Xinjiang Alaska grayling, broadly falling into dominant inheritance's labelling yet with these labellings, detection efficiency is high not as codominant markers such as microsatellite markers.It addition, the shortcoming of isoenzyme mark to be polymorphism low, the stability of RAPD labelling 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, being characterized in that kind is many, allele 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 stable.But owing to lacking Xinjiang Alaska grayling microsatellite marker, limit the development of its molecule genetics research, so in the urgent need to obtaining microsatellite marker to carry out research and the application of the aspects such as Xinjiang Alaska grayling genetic diversity, individual identification and genealogical identification.
Summary of the invention
It is an object of the invention to provide the Xinjiang Alaska grayling DNA molecular Genetic Markers of a kind of high polymorphism, i.e. the method for quick of Xinjiang Alaska grayling microsatellite marker Thymalag, to make up the deficiency of prior art.
The basic conception of the present invention is mainly by the micro-satellite core sequence contained in the Alaska grayling DNA sequence of Xinjiang, its design specific primers at both ends go forward side by side performing PCR detection, thus detecting the Xinjiang each individuality of the Alaska grayling hereditary variation in this micro-satellite district rapidly, obtain this primer (microsatellite marker Thymalag) the polymorphism collection of illustrative plates to Xinjiang Alaska grayling, 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, called after Thymalag from the Alaska grayling DNA sequence of Xinjiang, and this microsatellite marker Thymalag can be used for Xinjiang Alaska grayling analysis of genetic diversity and genealogical identification.
The present invention completes according to following operational aspect: first extract the genomic DNA in the Alaska grayling muscle of Xinjiang diluted for use；The micro-satellite core sequence contained in the Alaska grayling genomic dna sequence of recycling Xinjiang, in its sequence design specific primers at both ends；Then genomic DNA individual in these primer pair Xinjiang Alaska grayling difference proles or Xinjiang Alaska grayling group is used to carry out pcr amplification, PCR primer is carried out denaturing polyacrylamide gel electrophoresis detection, so that it is determined that the genotype of each individuality, it is thus achieved that the genetic polymorphism collection of illustrative plates of Xinjiang Alaska grayling.
Extracting Xinjiang Alaska grayling genomic DNA, diluted for adding 1 μ l in 100ng/ μ l, each PCR reaction, reaction cumulative volume is 25 μ l.
DNA sequence containing microsatellite sequence is:
1cccctgttacttgttggcgtaacaaagtgtgtgtgtttgtgtgtgtgtgtgtttgtgtgt
61gtttttctgtgtgcagggaaacacacttgttccttgacactgctttgaagacactttatg
121ttggggtttcctatattttggcagttacctgtagctatctgtaggaaattcctcacactt
181gcccattatcactttttaaattcaacctcctcttagtattttaatggacacaccatcatg
241atagattcatctctaacgagcaatagttaacagaaatacccttcctttattcaagccaaa
301ctctaaacagtttaaactatctatccagcttcactgcatctctgtttccac
Wherein micro-satellite core sequence is: TGTGTGTGTGTGTG
Thymalag micro-satellite primers sequence is:
Normal chain 5 '-CCCTGTTACTTGTTGGCGTAA-3 ',
Minus strand 5 '-TGGGCAAGTGTGAGGAATTT-3 ', uses annealing temperature during this primer to be 58 DEG C.
Micro-satellite core sequence and primer sequence are the cores of the present invention, present polymorphism in the Alaska grayling Diversity Detection of Xinjiang.
The application of sample parameter of pcr amplification is: it is 25 μ l that each PCR reacts cumulative volume, including 100ng Xinjiang Alaska grayling genomic DNA；10 × PCRBuffer, 2.5 μ l；Mg2+1.5mmol/L；Tag enzyme 1u；The each 0.1mmol/L of dNTP；The each 10pmol of above-mentioned primer；Add ddH2O to 25 μ l.The program parameter that PCR instrument is set during this primer is used to be: 94 DEG C of degeneration 2min；94 DEG C of 30sec, 58 DEG C of 45sec, 72 DEG C of 40sec, this reaction carries out 35 circulations；72 DEG C extend 5min, 4 DEG C of preservations.
The detecting step of PCR primer: PCR primer is easily 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 liquor colour developing 5min of 3%, finally soak 5min with the glacial acetic acid solution of 10%, the polymorphism collection of illustrative plates of Xinjiang Alaska grayling microsatellite marker Thymalag can be obtained.
Therefore, it is characteristic of the invention that the mapping genetic variations of the Thymalag microsatellite marker that can obtain Xinjiang Alaska grayling efficiently, method is easy.And, for other molecular genetic marker technique, microsatellite marker meets Mendelian inheritance pattern, and in codominant inheritance, acquired results can detect the genotype of each individuality of Xinjiang Alaska grayling intuitively；And it is applied in different proles or Xinjiang Alaska grayling group the structure etc. of genetic marker, genealogical identification and genetic map between individuality.
Accompanying drawing explanation
Fig. 1 is microsatellite marker Thymalag of the present invention to the individual detection collection of illustrative plates of Xinjiang Alaska grayling 20 20 individualities of Xinjiang Alaska grayling (numbering 1 20 be).
Detailed description of the invention
Describe the present invention by the examples below in detail in Xinjiang Alaska grayling Thymalag micro-satellite core sequence DNA molecular Genetic Markers method.First the genomic DNA in the Alaska grayling muscle of Xinjiang diluted for use are extracted；The micro-satellite core sequence contained in the Alaska grayling DNA sequence of recycling Xinjiang, in its sequence design specific primers at both ends；Then genomic DNA individual in these primer pair Xinjiang Alaska grayling difference proles or Xinjiang Alaska grayling group is used to carry out pcr amplification, PCR primer is carried out denaturing polyacrylamide gel electrophoresis detection, determine the genotype of each individuality, namely obtain the genetic polymorphism collection of illustrative plates of Xinjiang Alaska grayling.
1, the extraction of Xinjiang Alaska grayling genomic DNA: being added in Eppendorf pipe by 100 μ g Xinjiang Alaska grayling muscle, add the E.C. 3.4.21.64 5 μ l of cell pyrolysis liquid 500 μ l and 20mg/ml, shake up gently, 37 DEG C overnight.Then adding 600 μ l phenol chloroform isoamyl alcohol (25 24 1) mixed liquors in cracked sample, after rocking 20min, 12000rpm is centrifuged 10min, takes supernatant.Add phenol chloroform isoamyl alcohol mixed liquor, repeat aforesaid operations 3 times.Take supernatant again, add the dehydrated alcohol of 2 times of volume coolings and the sodium acetate of the 3mol/L of 1/10 volume, 12000rpm is centrifuged 10min, abandoning supernatant, retains DNA precipitation, then by this precipitation of 70% washing with alcohol 2 times, after ethanol volatilization completely, with TE buffer solution DNA, and dilute for 100ng/ μ l, 4 DEG C of preservations.
2, the design of micro-satellite primers: on Alaska grayling DNA sequence basis, Xinjiang, 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.Owing to micro-satellite core sequence mutation rate is of a relatively high, causing increase or the minimizing of microsatellite DNA core sequence number of repetition, the i.e. change of microsatellite DNA sequence length, this is the root of detection microsatellite polymorphism.The specific primer sequence at the core sequence two ends, micro-satellite district of the present invention is: normal chain 5 '-CCCTGTTACTTGTTGGCGTAA-3 ', minus strand 5 '-TGGGCAAGTGTGAGGAATTT-3 ', uses annealing temperature during this primer to be 58 DEG C.
3, pcr amplification: first application of sample, application of sample amount is as follows: Xinjiang Alaska grayling genomic DNA (100ng/L), 1 μ l；10 × PCRBuffer, 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 aquesterilisa to 25 μ l.Secondly, carrying out PCR reaction, its PCR amplification instrument program parameter is: 94 DEG C of degeneration 2min；94 DEG C of 30sec, 58 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: PCR primer be separated by electrophoresis on the denaturing polyacrylamide gel of 8%, electrophresis 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 liquor colour developing 5min of 3%, finally soak 5min with the glacial acetic acid solution of 10% and can obtain the Xinjiang Alaska grayling polymorphism collection of illustrative plates at the micro-satellite core sequence of Thymalag, as shown in Figure 1.Fig. 1 result shows, 20 individual coamplifications of Xinjiang Alaska grayling go out 8 allele, illustrate that microsatellite marker Thymalag has higher polymorphism, be appropriate to research and the application of the aspects such as Xinjiang Alaska grayling genetic diversity and hereditary constitution analysis, individual identification and genealogical identification.
<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 (Thymallusarcticusgrubei)
<220>
<221>repeat_region
<222>(38)...(51)
<400>1
1cccctgttacttgttggcgtaacaaagtgtgtgtgtttgtgtgtgtgtgtgtttgtgtgt
61gtttttctgtgtgcagggaaacacacttgttccttgacactgctttgaagacactttatg
121ttggggtttcctatattttggcagttacctgtagctatctgtaggaaattcctcacactt
181gcccattatcactttttaaattcaacctcctcttagtattttaatggacacaccatcatg
241atagattcatctctaacgagcaatagttaacagaaatacccttcctttattcaagccaaa
301ctctaaacagtttaaactatctatccagcttcactgcatctctgtttccac
Claims (5)
1. one group utilizes the microsatellite marker Thymalag primer that Xinjiang Alaska grayling hereditary variation is detected, it is characterised in that
Wherein said primer is sequence:
Normal chain 5 '-CCCTGTTACTTGTTGGCGTAA-3 ',
Minus strand 5 '-TGGGCAAGTGTGAGGAATTT-3 ', uses annealing temperature during this labelling to be 58 DEG C.
2. utilize the microsatellite marker Thymalag method that Xinjiang Alaska grayling hereditary variation is detected, it is characterised in that first extract the genomic DNA in the Alaska grayling muscle of Xinjiang diluted for use；The micro-satellite core sequence contained in the Alaska grayling DNA sequence of recycling Xinjiang, use specific primer that the genomic DNA of Xinjiang Alaska grayling Different Individual is carried out pcr amplification, PCR primer is carried out denaturing polyacrylamide gel electrophoresis detection, so that it is determined that the genotype that Xinjiang Alaska grayling Different Individual is in Thymalag core sequence district, wherein said Xinjiang Alaska grayling microsatellite marker Thymalag and specific primer be:
Normal chain 5 '-CCCTGTTACTTGTTGGCGTAA-3 ',
Minus strand 5 '-TGGGCAAGTGTGAGGAATTT-3 ', uses annealing temperature during this labelling to be 58 DEG C.
3. utilize the microsatellite marker Thymalag method that Xinjiang Alaska grayling hereditary variation is detected as claimed in claim 2, it is characterized in that, the pcr amplification that the genomic DNA that different Xinjiang Alaska grayling is individual is carried out, its application of sample parameter is: it is 25 μ l that each PCR reacts cumulative volume, including 100ng Xinjiang Alaska grayling genomic DNA；10 × PCRBuffer, 2.5 μ l；Mg2+1.5mmol/L；Taq enzyme 1u；dNTP0.1mmol/L；The each 10pmol of specific primer in claim 1；Finally add ddH2O to 25 μ l；The program parameter arranging PCR instrument is: 94 DEG C of degeneration 5min；94 DEG C of 45sec, 58 DEG C of 1min, 72 DEG C of 40sec, this reaction carries out 35 circulations；72 DEG C extend 5min, 4 DEG C of preservations.
4. utilize the microsatellite marker Thymalag method that Xinjiang Alaska grayling hereditary variation is detected as claimed in claim 2, it is characterized in that, PCR primer is carried out the step of denaturing polyacrylamide gel electrophoresis detection: PCR primer within 1.5 hours, be easily 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, 30min is soaked again with the silver nitrate solution of 0.1%, with the sodium carbonate liquor colour developing 5min of 3%, finally soak 5min with the glacial acetic acid solution of 10%.
5. the application on genetic polymorphism map construction between the Alaska grayling colony of Xinjiang of the method described in claim 2-4.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110042169A (en) * | 2019-05-31 | 2019-07-23 | 中国水产科学研究院黑龙江水产研究所 | A kind of T. grubii group specific molecular marker primer, kit and identification method |
| CN114982678A (en) * | 2022-07-21 | 2022-09-02 | 新疆额河生态养殖科技有限公司 | Artificial breeding operating system for fennel fish |
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| WO2007084499A2 (en) * | 2006-01-13 | 2007-07-26 | San Diego State University | Disease control in shrimp |
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| WO2007084499A2 (en) * | 2006-01-13 | 2007-07-26 | San Diego State University | Disease control in shrimp |
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| 刘云国等: "茴鱼微卫星标记开发及保护遗传学研究进展", 《水产科学》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110042169A (en) * | 2019-05-31 | 2019-07-23 | 中国水产科学研究院黑龙江水产研究所 | A kind of T. grubii group specific molecular marker primer, kit and identification method |
| CN110042169B (en) * | 2019-05-31 | 2022-11-18 | 中国水产科学研究院黑龙江水产研究所 | Molecular marker primer, kit and identification method for group specificity of Fennel fish in Heilongjiang |
| CN114982678A (en) * | 2022-07-21 | 2022-09-02 | 新疆额河生态养殖科技有限公司 | Artificial breeding operating system for fennel fish |
| CN114982678B (en) * | 2022-07-21 | 2024-02-23 | 新疆额河生态养殖科技有限公司 | Artificial breeding operation method for arctic fennel |
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