CN101736084A

CN101736084A - Methods for acquiring micro-satellite sequence and polymorphic micro-satellite markers of hucho taimen and polymorphic micro-satellite markers of hucho taimen

Info

Publication number: CN101736084A
Application number: CN200910073115A
Authority: CN
Inventors: 匡友谊; 佟广香; 尹家胜; 孙效文
Original assignee: Heilongjiang River Fisheries Research Institute of Chinese Academy of Fishery Sciences
Current assignee: Heilongjiang River Fisheries Research Institute of Chinese Academy of Fishery Sciences
Priority date: 2009-10-29
Filing date: 2009-10-29
Publication date: 2010-06-16
Anticipated expiration: 2029-10-29
Also published as: CN101736084B

Abstract

The invention discloses methods for acquiring a micro-satellite sequence and polymorphic micro-satellite markers of hucho taimen and the polymorphic micro-satellite markers of the hucho taimen, which relate to a method for acquiring the micro-satellite sequence and the polymorphic micro-satellite markers and the polymorphic micro-satellite markers. The method for acquiring the micro-satellite sequence comprises the following steps: firstly, extracting a hucho taimen genome DNA; secondly, performing enzyme cutting on the hucho taimen genome DNA and constructing a micro-satellite enrichment library; and thirdly, performing colony PCR amplification and performing positive clone detection and sequencing. The method for acquiring the polymorphic micro-satellite markers of the hucho taimen comprises the following steps: performing the first step to the third step the same as that of the method for acquiring the micro-satellite sequence; fourthly, analyzing the micro-satellite sequence of the hucho taimen and designing a primer; and fifth, performing polymorphic identification on the primer of the micro-satellite sequence. The method acquires seven pairs of the polymorphic micro-satellite markers of the hucho taimen. The biological materials obtained by the methods can be used for conservation genetics, genetic relationship analysis, linkage map construction and genetic management of cultured populations of the hucho taimen.

Description

The acquisition methods and the Zhe Luo salmon polymorphic micro-satellite markers of wise man's sieve salmon microsatellite sequence and polymorphic micro-satellite markers

Technical field

The present invention relates to the acquisition methods and the polymorphic micro-satellite markers of a kind of microsatellite sequence and polymorphic micro-satellite markers.

Background technology

Wise man sieve salmon is China original inhabitants' rare famous and precious fish, is the fish of build maximum in the salmon fishes.Because factors such as environmental degradation and overfishings, its present stock number is very rare, endangered; Therefore, wise sieve salmon was put into " Chinese animals on the brink of extinction Red Data Book (fish) " in 1998, was put into " the red register of Chinese species " in 2004.Wise man sieve salmon while also is a kind of good cold water cultured fishes, and it has, and growth is quick, the characteristics of strong stress resistance, has successfully carried out the artificial domestication of wild wise sieve salmon at present, and has successfully carried out mass-producing breeding and breed on this basis.But because its autotomy is serious, non-refractory, lower oxygen concentration resistance and in characteristics such as northern area low temperature season (as the winter-spring season water temperature in season below 10 ℃) poor growths not, culturing area and the cultured output of wise sieve salmon have been limited, increased aquaculture cost, therefore be necessary wise sieve salmon of present artificial domestication is carried out further seed selection.

Development along with biological technical field; molecule marker obtains competent development in the selection breeding of protection genetics, evolutionary genetics and the fish of species; especially the application in the selection breeding of animal; promoted the molecular marker assisted selection development of technology greatly, success just has the selection (patent No.: ZL 02821303.3) of tilapia luteotropin gene to salt-tolerance character in fish.Wise man sieve salmon is as endangered species and good breed kind; and available molecule marker quantity is very limited; the nucleotide sequence of wise sieve salmon of including among the Genbank is only more than 270 at present; 153 microsatellite sequences for the submission of laboratory, inventor place are wherein arranged; all the other major parts are the plastosome portion gene sequence of wise sieve salmon; only tens of available microsatellite molecular markers; therefore the microsatellite marker of developing wise sieve salmon is protected genetics to these species; the research of evolutionary genetics is very necessary, and the while also provides competent molecule marker for the molecular marker assisted selection of wise sieve salmon.

Little satellite is the dna sequence dna of short series connection repetition more than 3～6 times of being made up of 1～6 Nucleotide in the genome, utilize the conserved sequence design primer of tumor-necrosis factor glycoproteins both sides, develop microsatellite marker (microsatellite marker is made up of microsatellite sequence and microsatellite sequence primer two portions) with this.Because microsatellite marker polymorphism degree height and codominant inheritance calculate heterozygosity by it and can reflect intragroup variation preferably, so microsatellite marker are crucial for genetics research.The exploitation of microsatellite marker can be divided into for 3 steps: one, clone or isolate to contain from genome and lack series connection multiple dna sequence dna, i.e. microsatellite sequence; Two, the primer of design pcr amplification in the conserved sequence of the both sides of microsatellite sequence; Three, adopt suitable method to identify the polymorphism of designed primer.The exploitation of microsatellite marker is at present wasted time and energy, and main difficulty concentrates in the optimization of acquisition, design of primers and primer PCR amplification condition of microsatellite sequence.The existing several different methods of the separation of microsatellite sequence or clone, as the screening by hybridization of small segment genomic library, FIASCO (Fast Isolation by AFLP of Sequences), enrichment with magnetic bead method etc., because the enrichment with magnetic bead method has the higher characteristics of yield of positive colony rate and microsatellite sequence, so widely used at present, but this method need adopt isotropic substance (shortcoming has radioactivity) or vitamin H secondary hybridization (shortcoming toxicity is bigger) technology, increased the isolating the risk and cost of microsatellite sequence, so in use have limitation.

Summary of the invention

The objective of the invention is in order to solve that present employing enrichment with magnetic bead method is separated or clone's microsatellite sequence exists radioactivity or toxicity bigger, increased the problem of the isolating the risk and cost of microsatellite sequence, and the acquisition methods of a kind of wise sieve salmon microsatellite sequence that provides and polymorphic micro-satellite markers and Zhe Luo salmon polymorphic micro-satellite markers.

The present invention wise man sieve salmon microsatellite sequence obtains according to the following steps: one, extract wise sieve salmon genomic dna; Two, the enzyme of wise sieve salmon genomic dna is cut the structure with enriched microsatellite library; Three, colony PCR amplification and carry out that positive colony detects and order-checking promptly obtains wise sieve salmon microsatellite sequence.

The present invention wise man sieve salmon polymorphic micro-satellite markers obtains according to the following steps: one, extract wise sieve salmon genomic dna; Two, the enzyme of wise sieve salmon genomic dna is cut the structure with enriched microsatellite library; Three, colony PCR amplification and carry out that positive colony detects and order-checking obtains wise sieve salmon microsatellite sequence; Four, wise sieve salmon microsatellite sequence is analyzed and design of primers; Five, microsatellite sequence primer polymorphism is identified, obtains to have wise sieve salmon polymorphic micro-satellite sequence and primer thereof.

Totally 7 pairs of the present invention wise man sieve salmon polymorphic micro-satellite markers;

The sequence of microsatellite marker HtaCa69 is shown in SEQ ID NO:1, and its forward primer sequence is 5 '-GCAGGCTCTCGCACTAACA-3 ', and the reverse primer sequence is 5 '-CTGTCCCATTTGATGTCTGATAA-3 ';

The sequence of microsatellite marker HtaCa101 is shown in SEQ ID NO:2, and its forward primer sequence is 5 '-GTCGTTTGCCTCACCTCATA-3 ', and the reverse primer sequence is 5 '-CGTTACAGCCACATTCCTACAA-3 ';

The sequence of microsatellite marker HtaCa109 is shown in SEQ ID NO:7, and its forward primer sequence is 5 '-AGGGGATTCGGCTATTTCAC-3 ', and the reverse primer sequence is 5 '-CCTCTCATTGTGTTGGAGCA-3 ';

The sequence of microsatellite marker HtaCa172 is shown in SEQ ID NO:3, and its forward primer sequence is 5 '-AACCGTCCCCTAACCCAAT-3 ', and the reverse primer sequence is 5 '-TGCTTACCTCTCCCCAAAGT-3 ';

The sequence of microsatellite marker HtaCa183 is shown in SEQ ID NO:4, and its forward primer sequence is 5 '-TCTGAGCGTTTGTTGAATGTAA-3 ', and the reverse primer sequence is 5 '-GCCGAGCAGTGTGTGAGTTA-3 ';

The sequence of microsatellite marker HtaCa185 is shown in SEQ ID NO:5, and its forward primer sequence is 5 '-GCCGAGCAGTGTGTGAGTTA-3 ', and the reverse primer sequence is 5 '-TCTGAGCGTTTGTTGAATGTAA-3 ';

The sequence of microsatellite marker HtaCa203 is shown in SEQ ID NO:6, and its forward primer sequence is 5 '-AGCGGAAATAGCAGGAGGTT-3 ', and the reverse primer sequence is 5 '-GAATACCACAGCCCAGCATT-3 '.

The acquisition methods of the present invention wise man sieve salmon microsatellite sequence can obtain wise sieve salmon microsatellite sequence fast, easily; The acquisition methods of the present invention wise man sieve salmon polymorphic micro-satellite markers can obtain wise sieve salmon polymorphic micro-satellite markers fast, easily.

Combine the advantage of FIASCO and enrichment with magnetic bead method in the inventive method, avoided radio isotope or the vitamin H secondary hybridization evaluation risk that positive colony brought in the enrichment with magnetic bead method simultaneously, have low, the safe advantage of cost.The biomaterial that the inventive method obtained can be used for the heredity management of protection genetics, sibship analysis, linkage map structure and the cultured population of wise sieve salmon.

Description of drawings

Fig. 1 selects for use restriction enzyme Tru9I and Tsp509I to carry out enzyme to cut in the embodiment 12, the length that endonuclease bamhi connects back step 2 c recovery is the agarose gel electrophoresis figure of the pcr amplification product of 500～900bp.Fig. 2 is endonuclease bamhi and biotin labeled (CA) of restriction enzyme Sau3AI in the embodiment 12 ₁₆The bacterium colony PCR electrophorogram of the enriched microsatellite library that oligonucleotide probe makes up.Fig. 3 is endonuclease bamhi and biotin labeled (CA) of restriction enzyme Tsp509I in the embodiment 12 ₁₆The bacterium colony PCR electrophorogram of the enriched microsatellite library that oligonucleotide probe makes up.Fig. 4 is endonuclease bamhi and biotin labeled (CA) of restriction enzyme Tru9I in the embodiment 12 ₁₆The bacterium colony PCR electrophorogram of the enriched microsatellite library that oligonucleotide probe makes up.Fig. 5 is endonuclease bamhi and biotin labeled (CA) of restriction enzyme CviQI in the embodiment 12 ₁₆The bacterium colony PCR electrophorogram of the enriched microsatellite library that oligonucleotide probe makes up.Fig. 6 is endonuclease bamhi and biotin labeled (CA) of restriction enzyme BfaI in the embodiment 12 ₁₆The bacterium colony PCR electrophorogram of the enriched microsatellite library that oligonucleotide probe makes up.Fig. 7 is endonuclease bamhi and biotin labeled (CA) of restriction enzyme TaqI in the embodiment 12 ₁₆The bacterium colony PCR electrophorogram of the enriched microsatellite library that oligonucleotide probe makes up.Fig. 8 is endonuclease bamhi and biotin labeled (CAG) of restriction enzyme Sau3AI in the embodiment 12 ₁₆The bacterium colony PCR electrophorogram of the enriched microsatellite library that oligonucleotide probe makes up.Fig. 9 is that microsatellite marker is the detected result figure of HtaCa101 in the embodiment 13.Figure 10 is that microsatellite marker is the detected result figure of HtaCa69 in the embodiment 13.Figure 11 is that microsatellite marker is the detected result figure of HtaCa172 in the embodiment 13.Figure 12 is that microsatellite marker is the detected result figure of HtaCa183 in the embodiment 13.Figure 13 is that microsatellite marker is the detected result figure of HtaCa185 in the embodiment 13.Figure 14 is that microsatellite marker is the detected result figure of HtaCa203 in the embodiment 13.Figure 15 is that microsatellite marker is the detected result figure of HtaCa109 in the embodiment 13.

Embodiment

Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.

Embodiment one: present embodiment wise man sieve salmon microsatellite sequence obtains according to the following steps: one, extract wise sieve salmon genomic dna; Two, the enzyme of wise sieve salmon genomic dna is cut the structure with enriched microsatellite library; Three, colony PCR amplification and carry out that positive colony detects and order-checking promptly obtains wise sieve salmon microsatellite sequence.

Embodiment two: the difference of present embodiment and embodiment one is: adopt restriction enzyme single endonuclease digestion or double digestion wise man sieve salmon genomic dna in the step 2.Other step and parameter are identical with embodiment one.

Embodiment three: the difference of present embodiment and embodiment two is: step 2 branch following steps realize:

The enzyme of a, wise sieve salmon genomic dna is cut:

The wise man sieve salmon genomic dna enzyme system of cutting is 20 μ L, and cutting buffer, 0.2 μ L concentration by 2 μ L, 10 * enzyme is that BSA, 2.5U restriction enzyme, the 5 μ L concentration of 10ng/ μ L are that the wise sieve salmon genomic dna of 100ng/ μ L and the sterilization deionized water of surplus are formed; It is 4h that enzyme is cut incubation time;

B, connection:

Linked system is 40 μ L, forms by the sterilization deionized water of 20 μ L step a endonuclease bamhis, the sticking terminal double link joint of 2 μ L, 4 μ L10 * buffer, the T4DNA of 6weiss unit ligase enzyme and surplus, and the connection of in 4 ℃ of water-baths, spending the night; Its double center chain joint prepares according to the following steps: the equal-volume melting concn is single stranded oligonucleotide connecting joint A and the single stranded oligonucleotide connecting joint B of 10pmol/L, and 95 ℃ of sex change 10min at the uniform velocity are cooled to 10 ℃ through 4h then, promptly obtain double-stranded joint;

C, purpose fragment are obtained:

Do amplimer with single stranded oligonucleotide connecting joint A and increase, amplification reaction system is 25 μ L, is the MgCl of 25mmol/L by 3 μ L step b connection product, 1.5 μ L primers, 2.5 μ L, 10 * Buffer, 1.5 μ L concentration ₂, 2 μ L concentration are that the aseptic ultrapure water of the dNTP of 2.5mmol/L, Taq enzyme that 0.3 μ L concentration is 5U/ μ L and surplus is formed; The amplified reaction program is: 72 ℃ of 2min, and 94 ℃ of pre-sex change 5min, circulation is set to 94 ℃ of sex change 30s, 53 ℃ of annealing 30s, 72 ℃ of extension 1min, totally 14～20 circulations, 72 ℃ are extended 10min; Amplified production concentration is 1% agarose gel electrophoresis detection, and cutting glue recovery length with Promega PCR product recovery test kit is the dna fragmentation of 500～900bp;

The structure of d, enriched microsatellite library:

I, hybridization

Get the dna fragmentation that 12 μ L step c reclaim and place 95 ℃ of environment sex change 10min, join 68 ℃, volume then immediately and be in the hybridization solution of 38 μ L and obtain hybrid dna behind the hybridization 1h; Wherein the hybridization solution of 38 μ L is that biotin labeled probe, the 5 μ L concentration that contain tumor-necrosis factor glycoproteins of 10 μ mol/L are the single stranded oligonucleotide connecting joint A of 10pmol/L by 1.5 μ L concentration, 15 μ L, 20 * SSC, 0.5 μ L mass concentration is 10% SDS and 16 μ L ddH ₂O forms;

II, balance magnetic bead

In the 1.0mL centrifuge tube, add 100 μ L Streptavidin magnetic beads and 200 μ L washing lotion C washing 2 times, then centrifuge tube is placed on magnetic bead is adsorbed on the tube wall, the reject supernatant liquor, wash magnetic bead 3～5 times with 200 μ L washing lotion D again, add 150 μ L washing lotion D room temperatures afterwards and place, promptly obtain the balance magnetic bead; The pH value is that the concentration of 8.0 EDTA is that 1mmol/L, pH value are that the concentration of 8.0 Tris-Cl is that the concentration of 10mmol/L, NaCl is 2mmol/L among the washing lotion C; Washing lotion D is that the mass concentration with 6 * SSC liquid dilution is 0.1% SDS solution;

III, affine seizure

The hybrid dna that step I is obtained adds 25 ℃ of temperature bath 20min in the Step II balance magnetic bead, remove supernatant liquor then, wash magnetic bead 2 times for 25 ℃ with washing lotion D again, each 10min washs magnetic bead 2 times for 68 ℃ with washing lotion E afterwards, washs magnetic bead 2 times with washing lotion F again, then use 200 μ L, 0.1 * TE washing magnetic bead 2 times, add 30 μ L, 0.1 * TE again at 95 ℃ of sex change 10min, collect supernatant liquor then, promptly obtain containing the single stranded DNA fragment of tumor-necrosis factor glycoproteins; Wherein washing lotion E is that the mass concentration with 3 * SSC liquid dilution is 0.1% SDS solution, and washing lotion F is 6 * SSC liquid;

IV, pcr amplification contain the dna fragmentation of microsatellite sequence

The PCR reaction system is 25 μ L, is primer 0.5 μ L, Taq archaeal dna polymerase 0.5 μ L by the mixing PCR damping fluid 18 μ L, the single stranded oligonucleotide connecting joint A that include 4 kinds of dNTP, and the supernatant liquor 4 μ L that Step II I collects and the sterilized water of surplus are formed; The pcr amplification reaction program is: 94 ℃ of pre-sex change 5min, circulation is set to 94 ℃ of sex change 30s, 53 ℃ of annealing 30s, 72 ℃ of extension 1min, totally 20～30 circulations, and 72 ℃ are extended 10min, reclaim the test kit purifying with Promega PCR product and reclaim, remove unnecessary primer, dNTP and joint;

V, T-carrier connect and the clone

T-carrier ligation system is 10 μ L, is made up of the dna fragmentation that 2 * connection damping fluid, 1 μ L carrier pMD18-T vector and 4 μ L step IV purifying in the 5 μ L T-carrier commercial packages reclaim; Connect in contrast with T carrier self simultaneously, 4 ℃ of connections are spent the night; Use CaCl again ₂The competence bacillus coli DH 5 alpha of preparation transforms, and obtains the genome enriched microsatellite library, and will arrange in order in the single colony lift culture plate in the library.Other step and parameter are identical with embodiment two.

The present embodiment restriction endonuclease is bought from NEB company.Enzyme is cut incubation temperature referring to the restriction enzyme operation instruction.

The T4DNA ligase enzyme is available from U.S. Promega company.PMD18-T vector is available from precious biotechnology (Dalian) company limited (Dalian TaKaRa company).The competence bacillus coli DH 5 alpha is available from lid peaceful biotechnology (Beijing) company limited.

The medicine that uses in the present embodiment, reagent, enzyme, competent cell and plasmid etc. are all buied easily, if no particular requirement then concentration be that product marks concentration.Not marked operation steps is referring to the reagent operation instruction in the present embodiment.

25 ℃ of temperature are bathed and are made vitamin H and Streptavidin combination among the present embodiment steps d III.

Connect in the damping fluid among the present embodiment steps d V and comprise ligase enzyme.

Embodiment four: the difference of present embodiment and embodiment three is: the biotin labeled probe that contains tumor-necrosis factor glycoproteins is biotin labeled (CA) among the step 2 d I ₁₆Oligonucleotide probe or biotin labeled (CAG) ₁₆Oligonucleotide probe.Other step and parameter are identical with embodiment three.

Embodiment five: the difference of present embodiment and embodiment four is: restriction enzyme is restriction enzyme Tsp509I, restriction enzyme Tru9I, restriction enzyme CviQI, restriction enzyme BfaI, restriction enzyme Sau3AI or restriction enzyme TaqI among the step 2 a; The base sequence that uses its single stranded oligonucleotide connecting joint of restriction enzyme Tsp509I A in the step 2 is 5 '-CTCGTAGACTGCGTACC-3 ', the base sequence of single stranded oligonucleotide connecting joint B is 5 '-AATTGGTACGCAGTCTAC-3 '; The base sequence that uses restriction enzyme Tru9I, CviQI or its single stranded oligonucleotide connecting joint of BfaI A is 5 '-GACGATGAGTCCTGAG-3 ', the base sequence of single stranded oligonucleotide connecting joint B is 5 '-TACTCAGGACTCAT-3 '; The base sequence that uses its single stranded oligonucleotide connecting joint of restriction enzyme Sau3AI A is 5 '-GATCGTCGACGGTACCGAATTCT-3 ', the base sequence of single stranded oligonucleotide connecting joint B is 5 '-CAGCTGCCATGGCTTAAGAACTG-3 '; The base sequence that uses its single stranded oligonucleotide connecting joint of restriction enzyme TaqI A is 5 '-GACGATGAGTCCTGAG-3 ', the base sequence of single stranded oligonucleotide connecting joint B is 5 '-CGCTCAGGACTCAT-3 '.Other step and parameter are identical with embodiment four.

In the present embodiment the sticking end sequence of restriction enzyme Tsp509I be 5 '-AATT, it is 65 ℃ that enzyme Qie Wendu is hatched, the sticking end sequence of restriction enzyme Tru9I is 5 '-TA, it is 65 ℃ that enzyme Qie Wendu is hatched, the sticking end sequence of restriction enzyme CviQI is 5 '-TA, it is 37 ℃ that enzyme Qie Wendu is hatched, the sticking end sequence of restriction enzyme BfaI is 5 '-TA, it is 37 ℃ that enzyme Qie Wendu is hatched, the sticking end sequence of restriction enzyme Sau3AI is 5 '-GATC, it is 37 ℃ that enzyme Qie Wendu is hatched, the sticking end sequence of restriction enzyme TaqIGATC is 5 '-CG, it is 65 ℃ that enzyme Qie Wendu is hatched.

Embodiment six: the difference of present embodiment and embodiment five is: use universal primer M13+ or M13 in the step 3 ^-With the probe that contains tumor-necrosis factor glycoproteins be that primer carries out colony PCR amplification, the PCR reaction system is 15 μ L, is that 10 * Buffer, 0.9 μ L concentration are the MgCl of 25mmol/L in the forward primer of 10 μ mol/L, reverse primer that 0.6 μ L concentration is 10 μ mol/L, the 1.5 μ L Taq archaeal dna polymerase commercial packages by concentration ₂, 1.2 μ L concentration are that the aseptic deionized water of the dNTP of 2.5mmol/L, Taq archaeal dna polymerase that 0.12 μ L concentration is 5U/ μ L and surplus is formed; In order single bacterium colony is chosen with aseptic toothpick and to be carried out bacterium colony PCR in the reaction tubes, the PCR response procedures is 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ of extension 1min, totally 30 circulations, 72 ℃ are extended 5min, PCR reaction product concentration is 1% agarose gel electrophoresis detection, and the picking pcr amplification product is that the positive colony with obvious band of 200～750bp checks order.Other step and parameter are identical with embodiment five.

Embodiment seven: the difference of present embodiment and embodiment six is: select biotin labeled (CA) among the step 2 d I for use ₁₆Oligonucleotide probe, then step 3 pcr amplification primer is M13+ and (CA) ₁₀, perhaps M13-and (CA) ₁₀Select biotin labeled (CAG) among the step 2 d I for use ₁₆Oligonucleotide probe then step 3 pcr amplification primer is M13+ and (CAG) ₆, perhaps M13-and (CAG) ₆Other step and parameter are identical with embodiment six.

Embodiment eight: present embodiment wise man sieve salmon polymorphic micro-satellite markers obtains according to the following steps: one, extract wise sieve salmon genomic dna; Two, the enzyme of wise sieve salmon genomic dna is cut the structure with enriched microsatellite library; Three, colony PCR amplification and carry out that positive colony detects and order-checking obtains wise sieve salmon microsatellite sequence; Four, wise sieve salmon microsatellite sequence is analyzed and design of primers; Five, microsatellite sequence primer polymorphism is identified, obtains wise sieve salmon polymorphic micro-satellite markers.

Embodiment nine: the difference of present embodiment and embodiment eight is: step 2 branch following steps realize:

The enzyme of a, wise sieve salmon genomic dna is cut:

B, connection:

C, purpose fragment are obtained:

Do amplimer with single stranded oligonucleotide connecting joint A and increase, amplification reaction system is 25 μ L, is the MgCl of 25mmol/L by 3 μ L step b connection product, 1.5 μ L primers, 2.5 μ L, 10 * Buffer, 1.5 μ L concentration ₂, 2 μ L concentration are that the aseptic ultrapure water of the dNTP of 2.5mmol/L, Taq enzyme that 0.3 μ L concentration is 5U/ μ L and surplus is formed; The amplified reaction program is: 72 ℃ of 2min, and 94 ℃ of pre-sex change 5min, circulation is set to 94 ℃ of sex change 30s, 53 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 14～20 circulations, 72 ℃ are extended 10min; Amplified production concentration is 1% agarose gel electrophoresis detection, and cutting glue recovery length with Promega PCR product recovery test kit is the dna fragmentation of 500～900bp;

The structure of d, enriched microsatellite library:

I, hybridization

II, balance magnetic bead

III, affine seizure

IV, pcr amplification contain the dna fragmentation of microsatellite sequence

The PCR reaction system is 25 μ L, is primer 0.5 μ L, Taq archaeal dna polymerase 0.5 μ L by the mixing PCR damping fluid 18 μ L, the single stranded oligonucleotide connecting joint A that include 4 kinds of dNTP, and the supernatant liquor 4 μ L that Step II I collects and the sterilized water of surplus are formed; The pcr amplification reaction program is: 94 ℃ of pre-sex change 5min, and circulation is set to 94 ℃ of sex change 30s, 53 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 20～30 circulations, and 72 ℃ are extended 10min, reclaim the test kit purifying with Promega PCR product and reclaim, remove unnecessary primer, dNTP and joint;

V, T-carrier connect and the clone

T-carrier ligation system is 10 μ L, is made up of the dna fragmentation that 2 * connection damping fluid, 1 μ L carrier pMD18-T vector and 4 μ L step IV purifying in the 5 μ L T-carrier commercial packages reclaim; Connect in contrast with T carrier self simultaneously, 4 ℃ of connections are spent the night; Use CaCl again ₂The competence bacillus coli DH 5 alpha of preparation transforms, and obtains the genome enriched microsatellite library, and will arrange in order in the single colony lift culture plate in the library;

The biotin labeled probe that contains tumor-necrosis factor glycoproteins is biotin labeled (CA) among the step 2 d I ₁₆Oligonucleotide probe or biotin labeled (CAG) ₁₆Oligonucleotide probe;

Restriction enzyme is restriction enzyme Tsp509I, restriction enzyme Tru9I, restriction enzyme CviQI, restriction enzyme BfaI, restriction enzyme Sau3AI or restriction enzyme TaqI among the step 2 a; The base sequence that uses its single stranded oligonucleotide connecting joint of restriction enzyme Tsp509I A in the step 2 is 5 '-CTCGTAGACTGCGTACC-3 ', the base sequence of single stranded oligonucleotide connecting joint B is 5 '-AATTGGTACGCAGTCTAC-3 '; The base sequence that uses restriction enzyme Tru9I, CviQI or its single stranded oligonucleotide connecting joint of BfaI A is 5 '-GACGATGAGTCCTGAG-3 ', the base sequence of single stranded oligonucleotide connecting joint B is 5 '-TACTCAGGACTCAT-3 '; The base sequence that uses its single stranded oligonucleotide connecting joint of restriction enzyme Sau3AI A is 5 '-GATCGTCGACGGTACCGAATTCT-3 ', the base sequence of single stranded oligonucleotide connecting joint B is 5 '-CAGCTGCCATGGCTTAAGAACTG-3 '; The base sequence that uses its single stranded oligonucleotide connecting joint of restriction enzyme TaqI A is 5 '-GACGATGAGTCCTGAG-3 ', the base sequence of single stranded oligonucleotide connecting joint B is 5 '-CGCTCAGGACTCAT-3 ';

Use universal primer M13+ or M13 in the step 3 ^-With the probe that contains tumor-necrosis factor glycoproteins be that primer carries out colony PCR amplification, the PCR reaction system is 15 μ L, is the MgCl of 25mmol/L by 10 * Buffer, 0.9 μ L concentration in 0.6 μ L forward primer, 0.6 μ L reverse primer, the 1.5 μ L Taq archaeal dna polymerase commercial packages ₂, 1.2 μ L concentration are that the aseptic deionized water of the dNTP of 2.5mmol/L, Taq archaeal dna polymerase that 0.12 μ L concentration is 5U/ μ L and surplus is formed; In order single bacterium colony is chosen with aseptic toothpick and to be carried out bacterium colony PCR in the reaction tubes, the PCR response procedures is 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ of extension 1min, totally 30 circulations, 72 ℃ are extended 5min, PCR reaction product concentration is 1% agarose gel electrophoresis detection, and the picking pcr amplification product is that the positive colony with obvious band of 200～750bp checks order;

Select biotin labeled (CA) among the step 2 d I for use ₁₆Oligonucleotide probe, then step 3 pcr amplification primer is M13+ and (CA) ₁₀, perhaps M13-and (CA) ₁₀Select biotin labeled (CAG) among the step 2 d I for use ₁₆Oligonucleotide probe then step 3 pcr amplification primer is M13+ and (CAG) ₆, perhaps M13-and (CAG) ₆Other step and parameter are identical with embodiment eight.

The present embodiment restriction endonuclease is bought from NEB company.

Embodiment ten: present embodiment and embodiment eight or nines' difference is: step 4 is analyzed with design of primers wise sieve salmon microsatellite sequence and is realized according to the following steps:

1. according to step 2 restriction enzyme and the grouping of sticking terminal double link joint sequence;

2. adopt corresponding sticking terminal jointing sequence to carry out carrier and joint removal according to grouping, program thereby is dna sequencing polluted sequence batch treating tool (patent application publication number CN101149743);

3. carry out the tumor-necrosis factor glycoproteins screening to removing the sequence that is obtained behind carrier and the joint sequence with tandem repeat finder software, the start and end position of in the analytical results file of tandem repeat finder, searching tumor-necrosis factor glycoproteins, multiplicity, tumor-necrosis factor glycoproteins with perl language program repeat.pl, and the length of definite sequence, produce a csv file that comprises sequence number, sequence length, tumor-necrosis factor glycoproteins unit, multiplicity, tumor-necrosis factor glycoproteins starting position and final position, carry out the Batch Design of micro-satellite primers by the csv file;

4. sequence length and the tumor-necrosis factor glycoproteins of determining in 3. according to step begin, final position, obtain the length of little satellite flanking sequence, with its be divided into flanking sequence less than 30bp, flanking sequence less than 80bp and flanking sequence 3 classes greater than 80bp;

5. back two classes in the above-mentioned classification are set up the primer3 input file with the perl language according to the 3. middle csv file of setting up of step, call the primer3 primer-design software and carry out the batch design of primers.Other step and parameter are identical with embodiment eight or nine.

The present embodiment step 4 is 3. middle to be suffix .dat file by name with perl language program repeat.pl at the analytical results file of tandem repeat finder.

Embodiment 11: present embodiment and embodiment eight, nine or tens' difference is: step 5 microsatellite marker polymorphism is identified: carry out PCR, the PCR reaction system is 15 μ L, is that 10 * Buffer, 0.9 μ L concentration are the MgCl of 25mmol/L in the forward primer of 10 μ mol/L, reverse primer that 0.6 μ L concentration is 10 μ mol/L, the 1.5 μ L TaqDNA polysaccharase commercial packages by 0.6 μ L concentration ₂, 1.2 μ L concentration are that the aseptic ultrapure water of the dNTP of 2.5mmol/L, Taq archaeal dna polymerase that 0.12 μ L concentration is 5U/ μ L and surplus is formed; The PCR response procedures is 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 30s, annealing 30s, 72 ℃ of extension 1min, and totally 25～30 circulations, 72 ℃ are extended 5min, and annealing temperature is carried out the gradient screening between 48～65 ℃; Pcr amplification product concentration is that 1.5% agarose gel electrophoresis detects, and the amplified production concentration of selecting to have clear band is 10% native polyacrylamide gel electrophoresis detection polymorphism.Other step and parameter are identical with embodiment eight, nine or ten.

Embodiment 12: present embodiment wise man sieve salmon microsatellite sequence obtains according to the following steps: one, extract wise sieve salmon genomic dna; Two, the enzyme of wise sieve salmon genomic dna is cut the structure with enriched microsatellite library; Three, colony PCR amplification and carry out that positive colony detects and order-checking promptly obtains wise sieve salmon microsatellite sequence;

Wherein step 2 divides following steps to realize:

The enzyme of a, wise sieve salmon genomic dna is cut:

B, connection:

C, purpose fragment are obtained:

The structure of d, enriched microsatellite library:

I, hybridization

II, balance magnetic bead

III, affine seizure

IV, pcr amplification contain the dna fragmentation of microsatellite sequence

V, T-carrier connect and the clone

Use universal primer M13+ or M13 in the step 3 ^-With the probe that contains tumor-necrosis factor glycoproteins be that primer carries out colony PCR amplification, the PCR reaction system is 15 μ L, is that 10 * Buffer, 0.9 μ L concentration are the MgCl of 25mmol/L in the forward primer of 10 μ mol/L, reverse primer that 0.6 μ L concentration is 10 μ mol/L, the 1.5 μ L Taq archaeal dna polymerase commercial packages by 0.6 μ L concentration ₂, 1.2 μ L concentration are that the aseptic deionized water of the dNTP of 2.5mmol/L, Taq archaeal dna polymerase that 0.12 μ L concentration is 5U/ μ L and surplus is formed; In order single bacterium colony is chosen with aseptic toothpick and to be carried out bacterium colony PCR in the reaction tubes, the PCR response procedures is 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ of extension 1min, totally 30 circulations, 72 ℃ are extended 5min, PCR reaction product concentration is 1% agarose gel electrophoresis detection, and the picking pcr amplification product is that the positive colony with obvious band of 200～750bp checks order;

Select biotin labeled (CA) among the step 2 d I for use ₁₆Oligonucleotide probe, then step 3 pcr amplification primer is M13+ and (CA) ₁₀, perhaps M13-and (CA) ₁₀Select biotin labeled (CAG) among the step 2 d I for use ₁₆Oligonucleotide probe then step 3 pcr amplification primer is M13+ and (CAG) ₆, perhaps M13-and (CAG) ₆

The present embodiment restriction endonuclease is bought from NEB company.

Present embodiment is selected for use restriction enzyme Tru9I and Tsp509I to carry out enzyme and is cut, endonuclease bamhi connect length that back step 2 c reclaims be 500～900bp pcr amplification product agarose gel electrophoresis figure as shown in Figure 1, " M " swimming lane standard specimen is Marker among Fig. 1, " 1 " swimming lane standard specimen is cut for the Tru9I enzyme and is connected the back pcr amplified fragment, and " 2 " swimming lane standard specimen is cut for the Tsp509I enzyme and connected the back pcr amplified fragment.

The endonuclease bamhi of present embodiment restriction enzyme Sau3AI and biotin labeled (CA) ₁₆The bacterium colony PCR electrophorogram of the enriched microsatellite library that oligonucleotide probe makes up as shown in Figure 2; The endonuclease bamhi of restriction enzyme Tsp509I and biotin labeled (CA) ₁₆The bacterium colony PCR electrophorogram of the enriched microsatellite library that oligonucleotide probe makes up as shown in Figure 3; The endonuclease bamhi of restriction enzyme Tru9I and biotin labeled (CA) ₁₆The bacterium colony PCR electrophorogram of the enriched microsatellite library that oligonucleotide probe makes up as shown in Figure 4; The endonuclease bamhi of restriction enzyme CviQI and biotin labeled (CA) ₁₆The bacterium colony PCR electrophorogram of the enriched microsatellite library that oligonucleotide probe makes up as shown in Figure 5; The endonuclease bamhi of restriction enzyme BfaI and biotin labeled (CA) ₁₆The bacterium colony PCR electrophorogram of the enriched microsatellite library that oligonucleotide probe makes up as shown in Figure 6; The endonuclease bamhi of restriction enzyme TaqI and biotin labeled (CA) ₁₆The bacterium colony PCR electrophorogram of the enriched microsatellite library that oligonucleotide probe makes up as shown in Figure 7.

The endonuclease bamhi of present embodiment restriction enzyme Sau3AI and biotin labeled (CAG) ₁₆The bacterium colony PCR electrophorogram of the enriched microsatellite library that oligonucleotide probe makes up as shown in Figure 8.

It is in 200～750bp scope that present embodiment is chosen mushroom PCR pcr amplification product in the enriched microsatellite library and bacterium colony with obvious band carries out sequencing.

Embodiment 13: present embodiment wise man sieve salmon polymorphic micro-satellite markers obtains according to the following steps:

Step 1 is identical with embodiment 12 to step 3;

Step 4 is analyzed with design of primers wise sieve salmon microsatellite sequence and is realized according to the following steps:

5. back two classes in the above-mentioned classification are set up the primer3 input file with the perl language according to the 3. middle csv file of setting up of step, call the primer3 primer-design software and carry out the batch design of primers.

Step 5 microsatellite marker polymorphism is identified: carry out PCR, the PCR reaction system is 15 μ L, is the MgCl of 25mmol/L by 10 * Buffer, 0.9 μ L concentration in 0.6 μ L forward primer, 0.6 μ L reverse primer, the 1.5 μ L Taq archaeal dna polymerase commercial packages ₂, 1.2 μ L concentration are that the aseptic ultrapure water of the dNTP of 2.5mmol/L, Taq archaeal dna polymerase that 0.12 μ L concentration is 5U/ μ L and surplus is formed; The PCR response procedures is 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 30s, annealing 30s, 72 ℃ of extension 1min, and totally 25～30 circulations, 72 ℃ are extended 5min, and annealing temperature is carried out the gradient screening between 48～65 ℃; Pcr amplification product concentration is that 1.5% agarose gel electrophoresis detects, and the amplified production concentration of selecting to have clear band is 10% native polyacrylamide gel electrophoresis detection polymorphism.

Present embodiment is measured 906 sequences altogether, wherein contains 798 tumor-necrosis factor glycoproteinss by analysis, and the tumor-necrosis factor glycoproteins yield is 88.08%, obtains 918 microsatellite sequences altogether, and statistics is as shown in table 1.

Table 1

The 5. middle design of primers in batches of step 4 adopts Primer3 software default parameter, and flanking sequence is carried out design of primers greater than the part of 30bp, design 471 pairs of primers altogether, but the primer design rate is 84.3%.

15 pairs of the primer selections that present embodiment is designed are synthesized, and 10 pairs of primers can amplify band clearly through screening wherein, these 10 pairs of primers are carried out polymorphism identify have 7 pairs of primers to present polymorphism.

7 pairs of microsatellite sequences that primer increased that present polymorphism are respectively HtaCa69 (shown in SEQ IDNO:1), HtaCa101 (shown in SEQ ID NO:2), HtaCa172 (shown in SEQ ID NO:3), HtaCa183 (shown in SEQ ID NO:4), HtaCa185 (shown in SEQ ID NO:5), HtaCa203 (shown in SEQ ID NO:6) and HtaCa109 (shown in SEQ ID NO:7).

The forward primer sequence of microsatellite marker HtaCa69 is 5 '-GCAGGCTCTCGCACTAACA-3 ', and the reverse primer sequence is 5 '-CTGTCCCATTTGATGTCTGATAA-3 ', and the amplification annealing temperature is 55 ℃.

The forward primer sequence of microsatellite marker HtaCa101 is 5 '-GTCGTTTGCCTCACCTCATA-3 ', and the reverse primer sequence is 5 '-CGTTACAGCCACATTCCTACAA-3 ', and the amplification annealing temperature is 55 ℃.

The forward primer sequence of microsatellite marker HtaCa109 is 5 '-AGGGGATTCGGCTATTTCAC-3 ', and the reverse primer sequence is 5 '-CCTCTCATTGTGTTGGAGCA-3 ', and the amplification annealing temperature is 52 ℃.

The forward primer sequence of microsatellite marker HtaCa172 is 5 '-AACCGTCCCCTAACCCAAT-3 ', and the reverse primer sequence is 5 '-TGCTTACCTCTCCCCAAAGT-3 ', and the amplification annealing temperature is 60 ℃.

The forward primer sequence of microsatellite marker HtaCa183 is 5 '-TCTGAGCGTTTGTTGAATGTAA-3 ', and the reverse primer sequence is 5 '-GCCGAGCAGTGTGTGAGTTA-3 ', and the amplification annealing temperature is 60 ℃.

The forward primer sequence of microsatellite marker HtaCa185 is 5 '-GCCGAGCAGTGTGTGAGTTA-3 ', and the reverse primer sequence is 5 '-TCTGAGCGTTTGTTGAATGTAA-3 ', and the amplification annealing temperature is 60 ℃.

The forward primer sequence of microsatellite marker HtaCa203 is 5 '-AGCGGAAATAGCAGGAGGTT-3 ', and the reverse primer sequence is 5 '-GAATACCACAGCCCAGCATT-3 ', and the amplification annealing temperature is 52 ℃.

The pcr amplification system is 15 μ L, is the MgCl of 25mmol/L by 10 * Buffer, 0.9 μ L concentration in 0.6 μ L forward primer, 0.6 μ L reverse primer, the 1.5 μ L Taq archaeal dna polymerase commercial packages ₂, 1.2 μ L concentration are that the aseptic ultrapure water of the dNTP of 2.5mmol/L, Taq archaeal dna polymerase that 0.12 μ L concentration is 5U/ μ L and surplus is formed; The PCR response procedures is 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 30s, annealing 30s (annealing temperature is decided by primer), 72 ℃ of extension 1min, and totally 25～30 circulations, 72 ℃ are extended 5min.It is that 10% native polyacrylamide gel electrophoresis detects with concentration that PCR finishes the back, microsatellite marker be HtaCa101 detected result as shown in Figure 9, microsatellite marker be HtaCa69 detected result as shown in figure 10, microsatellite marker be HtaCa172 detected result as shown in figure 11, microsatellite marker be HtaCa183 detected result as shown in figure 12, microsatellite marker be HtaCa185 detected result as shown in figure 13, microsatellite marker be HtaCa203 detected result as shown in figure 14, microsatellite marker be HtaCa109 detected result as shown in figure 15.

According to detected result as can be known microsatellite marker HtaCa69, the HtaCa101, HtaCa172, HtaCa183, HtaCa185, HtaCa203 and the HtaCa109 that obtain of present embodiment all show polymorphism, can be used for the research of the aspects such as cultured population heredity management such as protection genetics, relationship analysis of wise sieve salmon.

The present embodiment method can obtain wise sieve salmon polymorphic micro-satellite sequence and primer thereof fast, easily.

Embodiment 14: totally 7 pairs of present embodiment wise man sieve salmon polymorphic micro-satellite markers;

Sequence table

＜110〉Heilongjiang Inst. of Aquatic Products, Chinese Academy of Aquatic Products Scie

＜120〉acquisition methods of wise sieve salmon microsatellite sequence and polymorphic micro-satellite markers and Zhe Luo salmon polymorphic micro-satellite markers

<160>29

<210>1

<211>350

<212>DNA

＜213〉wise sieve salmon (Hucho taimen)

<220>

＜223〉sequence of wise sieve salmon polymorphic micro-satellite markers HtaCa69.

<400>1

AGGGATGTTT?GTTAGCTTGT?TTATATTGGC?GGTATTGGAG?TAGAGTGATG?GGTAGAGGTG?60

AGAGGTCAAC?CAATGTAATG?AGGACGGGCT?GCTTATCACA?GTACACAACA?TGCAGGCTCT?120

CGCACTAACA?AACACATTGG?ACACACACAC?ACACACACAC?ACACACACAC?ACACACACAC?180

ACACACACAC?ACAGAAGTAG?GCAGCCAGAG?GGAGTTGGAG?CTATTGAAAA?GGGCAGCGTG?240

TGTTTACAGT?CAGATATCAG?AAAAGGTTAT?CAGACATCAA?ATGGGACAGC?CGTGTCTCAG?300

CCAATCACAC?AGAGGGACCA?GAGGCACGGT?AACGAATCAC?AGACAGACAG?350

<210>2

<211>892

<212>DNA

＜213〉wise sieve salmon (Hucho taimen)

<220>

＜223〉sequence of wise sieve salmon polymorphic micro-satellite markers HtaCa101.

<400>2

CTAAATAGCT?AACAAAATGG?CTACACTGAC?TATCTGCGTT?GACCCTTTTA?TTTTGCACTG?60

ACTATCACAG?GACTCCACAC?ACACACGCAC?ACACACACAC?ACACACACAC?ACACACACAC?120

ACACACACAC?ACACACACAC?ACACACTTAA?TACACTGGGG?AGGGACACCG?AGACAGAGGA?180

GCAATGAGAA?AGATACTGAA?AGTGGATGAG?ATAGAGAAGA?TGAGGAAGAC?AACATTTAGG?240

AGAGATAAGA?TGTGTGCTGA?TAATATTGAC?GGAAATATAT?GGTAATACAA?AGTAGTAAAA?300

GAGAGAGATA?AAGACGGCGA?TAGAGAGGGA?ATGAGAGACA?GAAGGAAGGA?AGGATGGTAA?360

AAGATTAATG?ATGAGGGGAT?GGCAGGCAAG?GTCAGGGCAG?AGCATGGAAG?AAAGGAGTGA?420

TGATGCAGCT?AGTGAACAAA?ACCTAAGGAC?AACAAGACTA?ACATGCTGGT?CAGGCACTCT?480

GTCCATACCA?CACTTAAAGG?ACAATGTGTC?CCCCAAGCCA?TACTAAACAA?TAGGACATAC?540

AGTCACAAAA?TAAACAGTGG?AATCTGAGGT?AAATAAGTGC?TGCTGTCAGT?GAGTGTATGA?600

TGATCTGAGT?GAATATCTAG?TCAAGTCCAT?GCACTATACA?GTATATCACA?CTATAGTGAC?660

ATTATCACAT?AGAAAGTTGT?ACCTTGGTCA?CATAAACAAA?CTGGCAATCT?CAAAGGGCTC?720

ATGTCGTTTG?CCTCACCTCA?TAACTTCTGT?AAAGGACAGT?AATGTGTATG?CACGCAAGTA?780

CACTTACACA?CACACACACA?CACACAGAGG?TTTATAGACC?ACTCTCCATC?AGTTTGGCAA?840

CCAGTTCTGA?TTGTCTTGTA?GGAATGTGGC?TGTAACGGCC?CTCCAGTGAA?TT?892

<210>3

<211>677

<212>DNA

＜213〉wise sieve salmon (Hucho taimen)

<220>

＜223〉sequence of wise sieve salmon polymorphic micro-satellite markers HtaCa172.

<400>3

ATTACTTATT?TATTGTACAA?TGACGGCCTA?CCCCGGCCAA?ACCGTCCCCT?AACCCAATTG?60

TGCGCCGTCC?TATGGGACTC?CGGATTACAG?CCAGTTTGTG?ATATAGCCCA?GGATTGAACC?120

AGGGTCTGTA?GTGACGCCTC?TAGCACTGAG?GTGCCATGCC?TTAGACCGCT?GCACCACTTG?180

GGAATGAATA?CATGGTTTAC?TCAGCTAATA?TTTAGGGCCA?AAATAGCATT?TCATGAAATG?240

TCAAAAATGT?AATTCTATAG?GAGCTTGTGT?GACACACGCT?CACTCACTCA?CGCACAGACA?300

CACTTGCGCG?CACACACACA?CACACACACA?CACACACACA?CACACACACA?CACACACACA?360

CAGAGGCAAA?GAGCACTGTG?AGTCCATTGA?GGATATGTGA?ACTGGCTCAT?TGACTTTGGG?420

GAGAGGTAAG?CATTCCGTTT?TTCCTCTACA?AGTTATACCC?TGATTTCTTT?GGAATGTGTT?480

TTAGCTTGTG?CAGGTGAGGG?CTGACAAATT?GTCTGTTGTA?TTTTGTTGCC?TTACATTCAA?540

CCGCAGATCA?TCAGTGATTA?CATAGTCCAT?GTACTTGACA?AAAGCTATCC?TGCTGTTTTG?600

TGAGTGGTCA?TGATTGAGGG?ATGCTTTGGG?GAATTTTGCT?GATAGTCTTG?ACCACCTGCT?660

ATTTGTTATG?TGCTGCC?677

<210>4

<211>408

<212>DNA

＜213〉wise sieve salmon (Hucho taimen)

<220>

＜223〉sequence of wise sieve salmon polymorphic micro-satellite markers HtaCa183.

<400>4

ATTCACACAT?GCGCGCCCAC?ACAGACACAC?ACACTTATGC?ACCCCTACAA?GCCAGGCGGA?60

TGTTCTTTGG?GTCAGCTATC?TTCTCTCAGA?GTGAATTTGG?ATGTCGTTTC?CTCTTGATTT?120

GTGTCGTGTC?TGAGCGTTTG?TTGAATGTAA?TTGTGTGGCA?CAAAGCTGTA?GACATCTCTC?180

TTTCTCTCTC?TCTCTCCCTC?CCACGGACAA?GCATCCCTGC?AGCTCACCCA?TAATTCCACA?240

GTTTCTATCT?CGACCATAAC?TCACACACTG?CTCGGCACTA?ACTAAAGGGG?CTGTCTGTAG?300

CGAGGCTGAT?TGCATGTTTG?TCCACCAAAT?TGTGGGTGCA?GCAGAGAGAG?ACTGCAGAGA?360

GGAAGAGAGT?ACACACACAG?CCTGGCCATG?CCAGGAGCCA?GTAGTGTT?408

<210>5

<211>430

<212>DNA

＜213〉wise sieve salmon (Hucho taimen)

<220>

＜223〉sequence of wise sieve salmon polymorphic micro-satellite markers HtaCa185.

<400>5

AACACTACTG?GCTCCTGGCA?TGGCCAGGCT?GTGTGTGTAC?TCTCTTCCTC?TCTGCAGTCT?60

CTCTCTGCTG?CACCCACAAT?TTGGTGGACA?AACATGCAAT?CAGCCTCGCT?ACAGACAGCC?120

CCTTTAGTTA?GTGCCGAGCA?GTGTGTGAGT?TATGGTCGAG?ATAGAAACTG?TGGAATTATG?180

GGTGAGCTGC?AGGGATGCTT?GTCCGTGGGA?GGGAGAGAGA?GAGAGAACGA?GAGATGTCTA?240

CAGCTTTGTG?CCACACAATT?ACATTCAACA?AACGCTCAGA?CACGACACAA?ATCAAGAGGA?300

AACGACATCC?AAATTCACTC?TGAGAGAAGA?TAGCTGACCC?AAAGAACATC?CGCCTGGCTT?360

GTAGGGGTGC?ATAAGTGTGT?GTGTCTGTGT?GGGCGCGCAT?GTGTGTGCTT?GTATGCGTGT?420

GTGTGTGAAT?430

<210>6

<211>568

<212>DNA

＜213〉wise sieve salmon (Hucho taimen)

<220>

＜223〉sequence of wise sieve salmon polymorphic micro-satellite markers HtaCa203.

<400>6

AATTGTTTAG?CGGAAATAGC?AGGAGGTTAT?CACATACTCT?GATGGTATCA?GTGTCACAAC?60

AACAAACTAC?TTGAAGAAAT?ACACACATCA?CACACACACA?CTCCCACGAA?CACAAATACT?120

CACACACTCC?AATGCTGGGC?TGTGGTATTC?CACGGGAGCA?ACTGAGAATA?CTGGGACTGT?180

TATCACATGC?TCATAGGAGC?GCACCGTGTG?GACAAAATAA?CATACACATG?CAGTATTCTG?240

TGCACACACA?CACACACACA?CACACACACA?CACACACACA?CACACACACA?CACACACACA?300

CACACAATCG?AACGAACACA?CACACACACA?CACACACATA?CATGCATGCA?CACACACACC?360

ATGGTGAGAC?TGTCCCACAG?CTTTAGCTGG?ACAACACAAA?CCACAGCAGA?AGTGGCAACA?420

GAGCACACAC?ACACACACTA?ACCGCAGTAC?TGTAACAGTG?TCTTATCCAT?GCTCCATGCA?480

GTCTTTGGGG?AGGTGTGGAC?CTAATGACTT?CTAATATCAG?CAAGCAGCCT?CTCCTCGCTT?540

GGTTGACACA?GACAACCCAG?TCCTAATT?568

<210>7

<211>493

<212>DNA

＜213〉wise sieve salmon (Hucho taimen)

<220>

＜223〉sequence of wise sieve salmon polymorphic micro-satellite markers HtaCa109.

<400>7

GTGTATAGTG?CAATGTCCCT?CTTAAATTTC?AAATGCTCCA?TATCCTTCCA?CCGTGTTTCC?60

CACATGTTCA?GACTACATTT?TGAGATAATA?CACAACATAT?ACAAAAGTAT?GTGGACACCC?120

CTTCAAATTA?AGGGGATTCG?GCTATTTCAC?CCACACCTGT?GTAGTTCCGT?ACATCGGTAA?180

AGAGACAGGT?GGGTCGGGGG?GGGCTAATAC?ACACACACAC?ACACACACAC?ACACTCACAC?240

ACAGACACAC?ACACACACAC?ACACCAGTCT?AACTTCTGTG?TGCTCCAACA?CAATGAGAGG?300

GTAACGGCTA?AACCAGCAGC?CTATTGGTCG?AAAAAACCCC?AAAAGGTTAA?CTTATAAACT?360

TGTGCGTAAT?GGCATGGCGT?TTCATATACG?CCACACTTCC?ACATGAAGTA?GACTTATAGG?420

CCTATGCTTA?TTGCCTTGTG?ACTGTAGATC?ACATTTTTAC?TGATACTGTA?GAACACTACA?480

GTACCATTGG?ATT?493

<210>8

<211>17

<212>DNA

＜213〉artificial sequence

<220>

＜223〉use its single stranded oligonucleotide connecting joint of restriction enzyme Tsp509I A sequence.

<400>8

CTCGTAGACT?GCGTACC 17

<210>9

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉use its single stranded oligonucleotide connecting joint of restriction enzyme Tsp509I B sequence.

<400>9

AATTGGTACG?CAGTCTAC 18

<210>10

<211>16

<212>DNA

＜213〉artificial sequence

<220>

＜223〉use restriction enzyme Tru9I, CviQI or its single stranded oligonucleotide connecting joint of BfaI A sequence.

<400>10

GACGATGAGT?CCTGAG?16

<210>11

<211>14

<212>DNA

＜213〉artificial sequence

<220>

＜223〉use restriction enzyme Tru9I, CviQI or its single stranded oligonucleotide connecting joint of BfaI B sequence.

<400>11

TACTCAGGAC?TCAT 14

<210>12

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉use its single stranded oligonucleotide connecting joint of restriction enzyme Sau3AI A sequence.

<400>12

GATCGTCGAC?GGTACCGAAT?TCT?23

<210>13

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉use its single stranded oligonucleotide connecting joint of restriction enzyme Sau3AI B sequence.

<400>13

CAGCTGCCAT?GGCTTAAGAA?CTG?23

<210>14

<211>16

<212>DNA

＜213〉artificial sequence

<220>

＜223〉use its single stranded oligonucleotide connecting joint of restriction enzyme TaqI A sequence.

<400>14

GACGATGAGT?CCTGAG?16

<210>15

<211>14

<212>DNA

＜213〉artificial sequence

<220>

＜223〉use its single stranded oligonucleotide connecting joint of restriction enzyme TaqI B sequence.

<400>15

CGCTCAGGAC?TCAT?14

<210>16

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the forward primer sequence of microsatellite marker HtaCa69.

<400>16

GCAGGCTCTC?GCACTAACA?19

<210>17

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the reverse primer sequence of microsatellite marker HtaCa69.

<400>17

CTGTCCCATT?TGATGTCTGA?TAA?23

<210>18

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the forward primer sequence of microsatellite marker HtaCa101.

<400>18

GTCGTTTGCC?TCACCTCATA?20

<210>19

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the reverse primer sequence of microsatellite marker HtaCa101.

<400>19

CGTTACAGCC?ACATTCCTAC?AA?22

<210>20

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the forward primer sequence of microsatellite marker HtaCa109.

<400>20

AGGGGATTCG?GCTATTTCAC?20

<210>21

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the reverse primer sequence of microsatellite marker HtaCa109.

<400>21

CCTCTCATTG?TGTTGGAGCA?20

<210>22

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the forward primer sequence of microsatellite marker HtaCa172.

<400>22

AACCGTCCCC?TAACCCAAT 19

<210>23

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the reverse primer sequence of microsatellite marker HtaCa172.

<400>23

TGCTTACCTC?TCCCCAAAGT?20

<210>24

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the forward primer sequence of microsatellite marker HtaCa183.

<400>24

TCTGAGCGTT?TGTTGAATGT?AA?22

<210>25

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the reverse primer sequence of microsatellite marker HtaCa183.

<400>25

GCCGAGCAGT?GTGTGAGTTA?20

<210>26

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the forward primer sequence of microsatellite marker HtaCa185.

<400>26

GCCGAGCAGT?GTGTGAGTTA?20

<210>27

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the reverse primer sequence of microsatellite marker HtaCa185.

<400>27

TCTGAGCGTT?TGTTGAATGT?AA?22

<210>28

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the forward primer sequence of microsatellite marker HtaCa203.

<400>28

AGCGGAAATA?GCAGGAGGTT?20

<210>29

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the reverse primer sequence of microsatellite marker HtaCa203.

<400>29

GAATACCACA?GCCCAGCATT?20

Claims

1. the acquisition methods of wise sieve salmon microsatellite sequence is characterized in that wise sieve salmon microsatellite sequence obtains according to the following steps: one, extract wise sieve salmon genomic dna; Two, the enzyme of wise sieve salmon genomic dna is cut the structure with enriched microsatellite library; Three, colony PCR amplification and carry out that positive colony detects and order-checking promptly obtains wise sieve salmon microsatellite sequence.

2. the acquisition methods of wise sieve salmon microsatellite sequence according to claim 1 is characterized in that adopting in the step 2 restriction enzyme single endonuclease digestion or double digestion wise man sieve salmon genomic dna.

3. the acquisition methods of wise sieve salmon microsatellite sequence according to claim 2 is characterized in that step 2 branch following steps realize:

The enzyme of a, wise sieve salmon genomic dna is cut:

B, connection:

Linked system is 40 μ L, forms by the sterilization deionized water of 20 μ L step a endonuclease bamhis, the sticking terminal double link joint of 2 μ L, 4 μ L10 * buffer, the T4 of 6weiss unit dna ligase and surplus, and the connection of in 4 ℃ of water-baths, spending the night; Its double center chain joint prepares according to the following steps: the equal-volume melting concn is single stranded oligonucleotide connecting joint A and the single stranded oligonucleotide connecting joint B of 10pmol/L, and 95 ℃ of sex change 10min at the uniform velocity are cooled to 10 ℃ through 4h then, promptly obtain double-stranded joint;

C, purpose fragment are obtained:

The structure of d, enriched microsatellite library:

I, hybridization

II, balance magnetic bead

III, affine seizure

IV, pcr amplification contain the dna fragmentation of microsatellite sequence

V, T-carrier connect and the clone

T-carrier ligation system is 10 μ L, is made up of the dna fragmentation that 2 * connection damping fluid, 1 μ L carrier pMD18-T vector and 4 μ L step IV purifying in the 5 μ L T-carrier commercial packages reclaim; Connect in contrast with T carrier self simultaneously, 4 ℃ of connections are spent the night; Use CaCl again ₂The competence bacillus coli DH 5 alpha of preparation transforms, and obtains the genome enriched microsatellite library, and will arrange in order in the single colony lift culture plate in the library.

4. the acquisition methods of wise sieve salmon microsatellite sequence according to claim 3 is characterized in that the biotin labeled probe that contains tumor-necrosis factor glycoproteins is biotin labeled (CA) among the step 2 d I ₁₆Oligonucleotide probe or biotin labeled (CAG) ₁₆Oligonucleotide probe.

5. the acquisition methods of wise sieve salmon microsatellite sequence according to claim 4 is characterized in that restriction enzyme is restriction enzyme Tsp509I, restriction enzyme Tru9I, restriction enzyme CviQI, restriction enzyme BfaI, restriction enzyme Sau3AI or restriction enzyme TaqI among the step 2 a; The base sequence that uses its single stranded oligonucleotide connecting joint of restriction enzyme Tsp509I A in the step 2 is 5 '-CTCGTAGACTGCGTACC-3 ', the base sequence of single stranded oligonucleotide connecting joint B is 5 '-AATTGGTACGCAGTCTAC-3 '; The base sequence that uses restriction enzyme Tru9I, CviQI or its single stranded oligonucleotide connecting joint of BfaI A is 5 '-GACGATGAGTCCTGAG-3 ', the base sequence of single stranded oligonucleotide connecting joint B is 5 '-TACTCAGGACTCAT-3 '; The base sequence that uses its single stranded oligonucleotide connecting joint of restriction enzyme Sau3AI A is 5 '-GATCGTCGACGGTACCGAATTCT-3 ', the base sequence of single stranded oligonucleotide connecting joint B is 5 '-base sequence of its single stranded oligonucleotide connecting joint of CAGCTGCCATGGCTTAAGAACTG-3 ' use restriction enzyme TaqI A is 5 '-GACGATGAGTCCTGAG-3 ', the base sequence of single stranded oligonucleotide connecting joint B is 5 '-CGCTCAGGACTCAT-3 '.

6. the acquisition methods of wise sieve salmon microsatellite sequence according to claim 5 is characterized in that in the step 3 with universal primer M13+ or M13 ^-With the probe that contains tumor-necrosis factor glycoproteins be that primer carries out colony PCR amplification, the PCR reaction system is 15 μ L, is that 10 * Buffer, 0.9 μ L concentration are the MgCl of 25mmol/L in the forward primer of 10 μ mol/L, reverse primer that 0.6 μ L concentration is 10 μ mol/L, the 1.5 μ L Taq archaeal dna polymerase commercial packages by 0.6 μ L concentration ₂, 1.2 μ L concentration are that the aseptic deionized water of the dNTP of 2.5mmol/L, Taq archaeal dna polymerase that 0.12 μ L concentration is 5U/ μ L and surplus is formed; In order single bacterium colony is chosen with aseptic toothpick and to be carried out bacterium colony PCR in the reaction tubes, the PCR response procedures is 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ of extension 1min, totally 30 circulations, 72 ℃ are extended 5min, PCR reaction product concentration is 1% agarose gel electrophoresis detection, and the picking pcr amplification product is that the positive colony with obvious band of 200～750bp checks order.

7. the acquisition methods of wise sieve salmon microsatellite sequence according to claim 6 is characterized in that selecting for use among the step 2 d I biotin labeled (CA) ₁₆Oligonucleotide probe, then step 3 pcr amplification primer is M13+ and (CA) ₁₀, perhaps M13-and (CA) ₁₀Select biotin labeled (CAG) among the step 2 d I for use ₁₆Oligonucleotide probe then step 3 pcr amplification primer is M13+ and (CAG) ₆, perhaps M13-and (CAG) ₆

8. the acquisition methods of wise sieve salmon polymorphic micro-satellite markers is characterized in that wise sieve salmon polymorphic micro-satellite markers obtains according to the following steps: one, extract wise sieve salmon genomic dna; Two, the enzyme of wise sieve salmon genomic dna is cut the structure with enriched microsatellite library; Three, colony PCR amplification and carry out that positive colony detects and order-checking obtains wise sieve salmon microsatellite sequence; Four, wise sieve salmon microsatellite sequence is analyzed and design of primers; Five, microsatellite sequence primer polymorphism is identified, obtains wise sieve salmon polymorphic micro-satellite markers.

9. the acquisition methods of wise sieve salmon polymorphic micro-satellite markers according to claim 8 is characterized in that step 4 is analyzed with design of primers wise sieve salmon microsatellite sequence to realize according to the following steps:

2. adopt corresponding sticking terminal jointing sequence to carry out carrier and joint removal according to grouping, program thereby is the dna sequencing polluted sequence batch treating tool;

5. back two classes in the above-mentioned classification are set up the primer3 input file with the perl language according to the 3. middle csv file of setting up of step, call the primer3 primer-design software and carry out the batch design of primers;

Step 5 microsatellite sequence primer polymorphism is identified: carry out PCR, the PCR reaction system is 15 μ L, is that 10 * Buffer, 0.9 μ L concentration are the MgCl of 25mmol/L in the forward primer of 10 μ mol/L, reverse primer that 0.6 μ L concentration is 10 μ mol/L, the 1.5 μ L Taq archaeal dna polymerase commercial packages by 0.6 μ L concentration ₂, 1.2 μ L concentration are that the aseptic ultrapure water of the dNTP of 2.5mmol/L, Taq archaeal dna polymerase that 0.12 μ L concentration is 5U/ μ L and surplus is formed; The PCR response procedures is 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 30s, annealing 30s, 72 ℃ of extension 1min, and totally 25～30 circulations, 72 ℃ are extended 5min, and annealing temperature is carried out the gradient screening between 48～65 ℃; Pcr amplification product concentration is that 1.5% agarose gel electrophoresis detects, and the amplified production concentration of selecting to have clear band is 10% native polyacrylamide gel electrophoresis detection polymorphism.

10. wise sieve salmon polymorphic micro-satellite markers is characterized in that totally 7 pairs of wise sieve salmon polymorphic micro-satellite markers;