CN102140449B - Method for screening scylla paramamosain microsatellite molecular marker - Google Patents
Method for screening scylla paramamosain microsatellite molecular marker Download PDFInfo
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Abstract
The invention relates to a method for screening a scylla paramamosain microsatellite molecular marker. The method comprises the following steps of: extracting scylla paramamosain genome DNA (Deoxyribonucleic Acid) and diluting the DNA for later use; designing a 5' anchor primer and performing PCR (Polymerase Chain Reaction) amplification; cloning an amplification product and sequencing; performing sequence analysis and designing a microsatellite specific marker; performing PCR amplification on the genome DNA of different geographical populations of scylla paramamosain or individuals in the populations by using the primer; detecting a PCR amplification product by using 6 percent denaturing polyacrylamide gel electrophoresis; and determining the genotype of each individual according to different migration distances of the amplification product to obtain a polymorphic map of the genetic variance of scylla paramamosain. The method is easy to operate, is safe and has a high positive rate and a true and reliable result. By adopting the method, the polymorphic map of the genetic variance of scylla paramamosain can be obtained quickly. The method is mainly applied to genetic variance analysis of scylla paramamosain, protection and management of the germ plasm resource, establishment of a genetic linkage map and genetic breeding.
Description
Technical field
The invention belongs to the microsatellite molecular marker of scylla paramamosain field, particularly relate to a kind of screening method of microsatellite molecular marker of scylla paramamosain.
Background technology
Microsatellite DNA (Microsatellite DNA) almost is present in all eukaryotic gene groups; The simple sequence of being made up of 1-6 Nucleotide repeats (Simple Sequence Repeats; SSR), be a kind of novel molecular labeling technique that grows up the nearly more than ten years.Little satellite has that quantity is many, stochastic distribution, polymorphum is high, repeatability is strong, be codominant inheritance and simple operation and other advantages, is widely used in the fields such as population genetic diversity analysis, plasm resource protection and management, genetic linkage maps structure and QTL location.
Chinese scholars has been developed microsatellite marker in most important aquatic animal.As: GUO etc. have developed 6 polymorphic microsatellite markers of large yellow croaker; Li Shao penta grade has been developed 19 microsatellite markers of Megalobrama amblycephala; Ma Hongyu etc. have reported 40 polymorphic microsatellite markers of verasper variegate and 31 polymorphic microsatellite markers of verasper moseri; Wang etc. have developed the polymorphic microsatellite marker in 17 EST sources of Pacific oyster; ELFSTROM etc. have developed 16 microsatellite markers of scallop; Yap etc. screen 8 polymorphic microsatellite markers of swimming crab; An etc. have screened 9 microsatellite markers of mitten crab.These polymorphic microsatellite markers have been widely applied in the researchs such as population genetic structural analysis, sibship evaluation, genetic linkage maps structure.
Scylla paramamosain (Scylla paramamosain) belongs to Arthropoda (Arthropoda), Crustachia (Crustacea), Decapoda (Decapoda), Portumidae (Portunidae), mud crab genus (Scylla); The torrid zone, subtropics and the temperate zone that mainly are distributed in the Pacific Ocean, the Indian Ocean are littoral; Mainly be distributed in the southeastern coast each province in China; Maximum with Fujian, Guangdong and Hainan, be one of important cultivated crabs class of China.In order to identify and protect the mud crab resource that extensively distributes in China, horse is ridden the waves and waits the plastosome to the mud crab of China's southeastern coast to study, the result confirm China extensively the mud crab of distribution be Scylla paramamosain, rather than the Young Crab of before having thought.At present, available Scylla paramamosain microsatellite marker quantity is considerably less, has seriously limited correlated inheritance and has learned the development of studying.
Summary of the invention
Technical problem to be solved by this invention provides a kind of screening method of microsatellite molecular marker of scylla paramamosain, and this method is simple to operate, safety, and positive rate is high, and real result is reliable, can obtain the polymorphum collection of illustrative plates of Scylla paramamosain heritable variation fast.Be mainly used in Scylla paramamosain Genetic Variation Analysis, plasm resource protection and management, genetic linkage maps structure and hereditary and selection aspect.
The screening method of a kind of microsatellite molecular marker of scylla paramamosain of the present invention comprises:
(1) extraction of Scylla paramamosain genomic dna and diluted for use;
(2) design 5 ' anchor primer and pcr amplification;
(3) clone of amplified production and order-checking;
(4) sequential analysis and little satellite special primer design;
(5) use primer that individual genomic dna in different geographical populations of Scylla paramamosain or the colony is carried out pcr amplification;
(6) denaturing polyacrylamide gel electrophoresis of use 6% detects pcr amplification product;
(7) confirm the genotype that each is individual according to the different migration distances of amplified production, obtain the microsatellite marker of 18 polymorphums altogether, thereby obtain the polymorphum collection of illustrative plates of Scylla paramamosain heritable variation.
The extraction of the Scylla paramamosain genomic dna in the said step (1) may further comprise the steps: a small amount of muscle tissue 100-150mg of clip Scylla paramamosain (being collected in Ningde City offshore sea waters, Fujian Province); Place shredding of 500 μ L tissue extracts; The adding final concentration is that Proteinase K and the final concentration of 20mg/mL is the RNaseA of 100 μ g/mL, and 55 ℃ digested 2-3 hour; The use volume ratio is 25: 24: 1 a phenol: chloroform: twice of primary isoamyl alcohol mixed solution extracting; Use the absolute ethyl alcohol deposit D NA of 2 times of volumes then, after 70% washing with alcohol and seasoning, be dissolved among the TE; Be 100ng/ μ L with the DNA concentration dilution at last, and be kept at-20 ℃ subsequent use.
Design 5 ' anchor primer in the said step (2) may further comprise the steps: design contains the primer that annexs base and repeat base; Wherein annex base portion and have the grappling effect; Prevent primer and template combine slide; Repeating base portion is that 2 bases repeat for 6 times, 3 or 4 bases repeat for 4 times, is used for combining with the repetition base of genomic dna; The primer that adopts has PCT6:5 '-KKVRVRV (CT)
6-3 ', PGT6:5 '-KKRVRVR (GT)
4-3 ', PCAC4:5 '-KKBDDBD (CAC)
4-3 ';
Described pcr amplification may further comprise the steps: each 5 ' anchor primer is that template is carried out pcr amplification with the Scylla paramamosain genomic dna, and reaction system is 25 μ L, comprises that genomic dna template 1 μ L, 5 ' anchor primer final concentration are 0.8 μ mol/L, Mg
2+Final concentration is that 1.5mmol/L, dNTP final concentration are 0.2mmol/L, Taq archaeal dna polymerase 0.75U, 1 * PCR buffer, and replenishing sterilization distilled water to TV at last is 25 μ L; Response procedures is: 94 ℃ of preparatory sex change 5 minutes, and the annealing temperature annealing of 94 ℃ of sex change 30 seconds, primer specific was extended 30 circulations 50 seconds for 50 seconds, 72 ℃; Extended 7 minutes in 72 ℃ at last; Agarose gel electrophoresis with 1.5% detects preparatory amplified production, and reclaims the amplified production of size between 250-800bp.
Clone and order-checking in the said step (3) may further comprise the steps: at first be that the PCR product that reclaims is connected in the pMD19-T carrier (TaKaRa), be transformed into then among the competent escherichia coli cell DH5 α, be inoculated into LB (Amp
+) grow in the substratum, utilize carrier universal primer and pcr amplification method to identify positive colony again, picking inserts clip size and utilizes the ABI3730 automatic DNA sequencer DNA to check order at the positive colony of 250-1000bp.
Sequential analysis in the said step (4) and the design of little satellite special primer may further comprise the steps: at first utilize 4.0 pairs of all sequences that obtained of biological software DNAMAN to compare, remove identical sequence; Utilize biological software SSRHUNTER 1.3 to search then and contain little satellite core repeating sequences; Utilize these microsatellite sequences of BLAST method search in the GenBank DB, whether to have identical sequence again, and remove those identical sequences; The microsatellite sequence that utilizes 5.0 couples of biological software Primer Premier to contain complete flanking region at last carries out design of primers;
Wherein, described microsatellite molecular marker of scylla paramamosain and corresponding special primer sequence thereof, as follows:
Scypa01
1 tccctaccta?ccattacacc?ccattaccca?ttccccattg?cccttctgtt?catcttacct
61 gccctaatct?gtaacctgtc?actttttttc?gttgtctcgt?tgctcctcct?cctcttcctc
121 ctcctgtact?attccatctc?catcaaatag?ctacaatatt?tattcattta?gattatttac
181 ttcccaaaca?gccatatcag?ggactacaga?tctgttgctg?tctggctgtc?ctttgtaata
The special primer sequence of Scypa01 mark:
F：TCCCTACCTACCATTACACCC
R：TATTACAAAGGACAGCCAGACA
Scypa02
1 tctgtaatca?gaccaaggag?gttttgtgtg?cagcagcagc?agcagcagca?gcagcagcag
61 cagcagcaac?acaccatatg?tactctcttg?agttctatga?aatcctgtta?tccactgttt
121 gattatcaca?aaaacttaag?tgtaatggat?agtgtaaaag?tgatctacaa?ctacttactg
181 taattgaatg?taatgcattt?tacttaaaga?aattaattga?gcaaaataac?acaagagatt
241 atcttattct?tagcttccag?tatggctatt?ttg
The special primer sequence of Scypa02 mark:
F：TCTGTAATCAGACCAAGGAGGT
R：CAAAATAGCCATACTGGAAGC
Scypa03
1 gcggttcatt?tgcttcgaaa?cagctaaagg?ggtttgtata?ttgcgttttg?tgtagtttgg
61 agtgcctgaa?ggcttattgc?ttggttcttc?gtagttgtag?tagtggttag?tagtggtggt
121 agtagtagaa?acacagaaaa?agtagtagta?gtagtagtag?tagtaattca?cacatatgat
181 agttctgcat?gtctagttgc?gtaaatgaca?ataaattaaa?tgactcaaga?ataataagga
241 caacccagtc?tcg
The special primer sequence of Scypa03 mark:
F：GCGGTTCATTTGCTTCG
R：CGAGACTGGGTTGTCCTTA
Scypa04
1 ccactcctgc?catcctcatt?actacaatca?tcacaaccac?cacctctctt?cctccatctc
61 cactacctcc?tcctctctta?ccgccaccac?taccacctct?cctcctcctc?ctcctcctcc
121 tcctgctcct?cctgcttctg?ctcgtcctac?tgctgctgct?gctgctccac?cacgaccacc
181 acaacccatg?catcatcact?gggtggctgc?gacaggcggg?atgggactca?cttacccctc
241 actgggacaa?agatgccgct?ggg
The special primer sequence of Scypa04 mark:
F：CCACTCCTGCCATCCTCATT
R：CCCAGCGGCATCTTTGTC
Scypa05
1 ggatagttgc?tggttgatga?agcctggaat?tgtggtagtc?atcagaatag?tagtagtagt
61 agtagttgta?gtagttgcaa?ttttctacta?ctaatactaa?tactactata?actactacta
121 ttactattac?tatcaccact?atttacctgc?atgcaaagcc?gcttcaatta?acattctacc
181 acaattcgcc?gcagacccc
The special primer sequence of Scypa05 mark:
F：GGATAGTTGCTGGTTGATGAAG
R：GGGGTCTGCGGCGAAT
Scypa06
1 tgagagaggc?agggtaggaa?aacaaagaaa?gagagaagga?agtaaaagat?gaaaggaggg
61 aagaaaaaac?gaaggagaga?aggaaaaaag?gaagaccagt?tgagtgtaag?taacgagagg
121 agacttgtgt?taagtaatgt?tttgttgtaa?ctcgctgctg?taatacttgt?actgatgaca
181 atagcgttta?ttattattat?tattattatt?attattatta?ttattatttg?tcttattatt
241 atctttattg?ttatatatgt?gtaatggaag?ttaacgcttt
The special primer sequence of Scypa06 mark:
F：TGAGAGAGGCAGGGTAGGAA
R：AAAGCGTTAACTTCCATTACACA
Scypa07
1 gcgctctgag?gcaagaagag?aaactaacca?gtaatagtag?aagagcacac?acacacacac
61 acacacacac?acacacacat?actcataggg?gaagggcagc?gagtcaaggc?ttcacgtgct
121 cgaatgaaag?ctttggtact?caccaggatc?ttgtttcatc?acctcttttc?ttatttcttc
181 ctttttcacc?cttttttttt?tgtaggagga?aagggtgatg?ggagatgtat?ggctaagc
The special primer sequence of Scypa07 mark:
F：GCGCTCTGAGGCAAGAAG
R：GCTTAGCCATACATCTCCCAT
Scypa08
1 acacgagaca?gagggaggct?cctgtactac?acatcactca?gggaagagag?agaagtaaat
61 ctttggggta?aaaacagtac?cagaaactcc?gtaacttgac?tgtaactttg?agctcataaa
121 gttttcaagg?aagttgtact?ctctctctct?ctctctctag?atggattaga?acacagaatc
181 ttgtatctcg?aacccgg
The special primer sequence of Scypa08 mark:
F：ACACGAGACAGAGGGAGGC
R：CCGGGTTCGAGATACAAGAT
Scypa09
1 cgagactcag?atcggatgat?ggtgatgaag?cacgaggaat?gcatgttgat?ctggggcgtg
61 gagggctgtg?atggtggtgg?tggtgatggt?ggtgggagta?gtagtagtag?tagtagtaat
121 agttgaggtt?attgttgttg?cagtcgtggt?agtagtagta?gtagtagtaa?tagtagtagt
181 aacagcagta?acaattgtaa?tcactgctag?tagcgg
The special primer sequence of Scypa09 mark:
F：CGAGACTCAGATCGGATGATG
R：CCGCTACTAGCAGTGATTACAAT
Scypa10
1 tagttctttc?ttccttctac?cgataccacc?accaccacca?ccacccccaa?aaccacaaga
61 gcaccacacc?ttaaaatacc?agcagcagta?acattagtag?ctctgttagc?actgatagca
121 aagacaatgg?agggaaaaaa?aaaaaaaaca?gaataagaaa?cttgtcaagg?caagaggtga
181 ggtggtcagc?caggcaggtg
The special primer sequence of Scypa10 mark:
F：TAGTTCTTTCTTCCTTCTACCGAT
R：CACCTGCCTGGCTGACC
Scypa11
1 aacgctacat?catactgcgt?acttcccagt?ggtgcacaca?cacacacaca?cacacacaca
61 cacacacacc?ataagtggat?ggataagtga?gtatctatgg?attacttatg?aaaatgagag
121 cataaagtat?tcttgttttt?gtagtggtgg?tggtgaagta?tttatagaag?tagtagtcgt
181 agtagtagta?gtaatatagt?agtagaagta?agcagaaata?gcaacag
The special primer sequence of Scypa11 mark:
F：AACGCTACATCATACTGC
R：CTGTTGCTATTTCTGCTT
Scypa12
1 accgccattc?attccattca?cccaccgttt?tgaatttaca?agtttattat?gaagaaaacg
61 caaagtacag?aagtcacgga?ggacagacag?acagacagac?agacagacag?actttacacg
121 caggcagggt?aactgaagga?ggccagacgc?aggtatagtg?gggctcggct?gttccttta
The special primer sequence of Scypa12 mark:
F：ACCGCCATTCATTCCATT
R：TAAAGGAACAGCCGAGCC
Scypa13
1 agcgtctgtc?cacccttagt?acccttgtac?cctcacccct?atgcacccca?atgtaccata
61 ccctgtgccc?ttcacctata?tattctctta?ctccctcctc?gtgcatttca?acgccccact
121 ctccctctcc?ccttgccata?caccacgtgt?gatgctctgg?cgttgtgcac?gtggaaggga
181 gcgtgctctg?agcgttggga?tacccgagag?gaggaggagg?aggaggagga?ggaaggagga
241 gcaggaggag?gaggagaagg?aggaggagaa?tacgaggttg?tgggaaagtc
The special primer sequence of Scypa13 mark:
F：AGCGTCTGTCCACCCTTAG
R：GACTTTCCCACAACCTCGTAT
Scypa14
1 gacccgattt?gcatctttag?tttttttttt?ttttttatgc?ataaagaatg?ccttccaaaa
61 caacaacaac?aacaacaaca?acaaaagtgg?gataaacaaa?aagcccacta?gtttgcctct
121 tcccagaatt?aaaaaaaaaa?aaaaaaacgg?gacaagatca?ccgaaacgga?agcc
The special primer sequence of Scypa14 mark:
F：CCCGATTTGCATCTTTAGTT
R：CTTCCGTTTCGGTGAT
Scypa15
1 ccgcgaaaca?aggagaacat?ttaagggttc?tttcttagta?cagggaataa?agcaggtgtg
61 gtgatgggtg?tgtggtgcag?cggttgttgt?ggtgtatgtt?agtggtggtg?tggtggtgtg
121 tttgcgtggt?gcatgaggga?gtgctttgtg?tgtgtgtgtg?tgtgtgtgtg?tgtgtgtgtg
181 tgtgtgtgtg?tgtgtgtata?gtttgatgga?tgctacaccg
The special primer sequence of Scypa15 mark:
F：GCGAAACAAGGAGAAC
R：GTGTAGCATCCATCAAAC
Scypa16
1 ctgcctcttt?aatgttgact?cctcactatt?ttatttattt?ttgttttgtt?ttaccagtga
61 attttagaat?gtctacccat?ttctttttgt?tgtattgatt?gttgttgttg?ttgttgttgt
121 tgttgttgct?tctgctacta?ctactaatac?taataccact?acatcttttg?tggtag
The special primer sequence of Scypa16 mark:
F：CTGCCTCTTTAATGTTGACTCC
R：CTACCACAAAAGATGTAGTGGTATT
Scypa17
1 taactggttg?gtcggctgat?tgaaagtgct?ggagaaaacg?gaataatttt?acgccgcagg
61 acgaaaagaa?gtgattggat?caggaatctg?aatcgaagag?gaaagatact?actgtagcta
121 agagagagag?agagagagag?agagagagag?agagagattt?ttttttccct?cagtatacac
181 tctaatggaa?aaataataac?agtactaaca?cacagaactc?agcttacgga?cgactc
The special primer sequence of Scypa17 mark:
F：ACTGGTTGGTCGGCTGATTG
R：GTCGTCCGTAAGCTGAGTTC
Scypa18
1 accaaaaaca?agaaatatca?ccactatcat?cattattcct?accacaacaa?caacaacaac
61 aacaaaacca?cgcccacagg?aaccagccac?caaaacctcc?ccaaaaaaac?acccaccaaa
121 atatattaaa?accgatacac?atatatatac?gtatacatcc?cctgggcagc?ctcacatatt
181 tttcctcttc?ccttccgttt?cctcccagag?cgacaaattt?tggtggcgaa?ggagacggta
The special primer sequence of Scypa18 mark:
F：CAAAAACAAGAAATATCACCA
R：CCGTCTCCTTCGCCA
Of the present invention is little satellite nucleotide sequence and primer sequence, and wherein Scypa01 is that 240 Nucleotide, Scypa02 are that 273 Nucleotide, Scypa03 are that 263 Nucleotide, Scypa04 are that 264 Nucleotide, Scypa05 are that 199 Nucleotide, Scypa06 are that 280 Nucleotide, Scypa07 are that 238 Nucleotide, Scypa08 are that 197 Nucleotide, Scypa09 are that 216 Nucleotide, Scypa10 are that 200 Nucleotide, Scypa11 are that 227 Nucleotide, Scypa12 are that 179 Nucleotide, Scypa13 are that 290 Nucleotide, Scypa14 are that 174 Nucleotide, Scypa15 are that 220 Nucleotide, Scypa16 are that 176 Nucleotide, Scypa17 are that 236 Nucleotide, Scypa18 are 240 Nucleotide.
Use primer in the said step (5) is 25 μ L to the reaction system that individual genomic dna in different geographical populations of Scylla paramamosain or the colony carries out pcr amplification, comprises that genomic dna template 1 μ L, upstream and downstream primer final concentration are 0.4 μ mol/L, Mg
2+Final concentration is that 1.5mmol/L, dNTP final concentration are 0.2mmol/L, Taq archaeal dna polymerase 0.75U, 1 * PCR buffer, and replenishing sterilization distilled water to TV at last is 25 μ L; Response procedures is: 94 ℃ of preparatory sex change 5 minutes, and the annealing temperature annealing of 94 ℃ of sex change 30 seconds, primer specific was extended 35 circulations 50 seconds for 50 seconds, 72 ℃; Extended 7 minutes in 72 ℃ at last.
The denaturing polyacrylamide gel electrophoresis of the use 6% in the said step (6) detects pcr amplification product: after 95 ℃ of sex change of PCR product, the about 3 μ L of last appearance carry out electrophoresis in 6% polyacrylamide gel; Obtained the PCR product electrophoretic image of Scylla paramamosain after dyed, the colour developing.
Described microsatellite marker is applied to fields such as Scylla paramamosain Genetic Variation Analysis, plasm resource protection and management, genetic linkage maps structure and marker-assisted breeding.
The present invention includes three kinds of enriched microsatellite libraries of Scylla paramamosain (CT)
n, (GT)
n, (CAC)
nStructure; The evaluation of positive colony and order-checking; The design of sequential analysis and micro-satellite primers; Final 18 amplified bands microsatellite marker of polymorphum clearly that obtains is used for the genetic analysis of Different Individual in different geographical populations of Scylla paramamosain or the colony with these 18 polymorphism marks, obtains the polymorphum collection of illustrative plates of the heritable variation of Scylla paramamosain.
Beneficial effect
(1) advantage such as simple to operate, quick, accurate, the sensitivity of method of the present invention can be accomplished a process in one week, has improved the efficient of screening microsatellite marker greatly;
(2) the present invention prepares the microsatellite marker of Scylla paramamosain simply, efficiently; Once can obtain hundreds of even thousands of microsatellite sequences; And the most microsatellite sequence that is obtained all can design primer, and the present invention is mainly used in fields such as Scylla paramamosain Genetic Variation Analysis, plasm resource protection and management, genetic linkage maps structure and marker-assisted breeding.
Description of drawings
The primer Scypa01 of Fig. 1 screening is to the detection collection of illustrative plates of Scylla paramamosain 25 individuals, and wherein M is a molecular weight standard, and 1-25 is that Scylla paramamosain is individual.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.Should be understood that in addition those skilled in the art can do various changes or modification to the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Embodiment 1
1, the extraction of Scylla paramamosain genomic dna
The a small amount of muscle tissue (about 100-150mg) of clip Scylla paramamosain (being collected in Ningde City offshore sea waters, Fujian Province), place 500 μ L tissue extracts (10mmol/L Tris-Cl, pH 8.0; 100mmol/L EDTA, pH8.0; 100mmol/L NaCl; Shred 0.5%SDS), add Proteinase K (final concentration is 20mg/ml) and RNaseA (final concentration is 100 μ g/mL), abundant mixing, 55 ℃ of digestion extremely clarification in 2-3 hour; Use phenol: chloroform: twice of primary isoamyl alcohol mixed solution (volume ratio is 25: 24: 1) extracting; Use the absolute ethyl alcohol deposit D NA of 2 times of volumes then, after 70% washing with alcohol and seasoning, be dissolved in 50 μ L TE (10mmol/L Tris-HCl, pH 8.0; 10mmol/L EDTA, pH 8.0) in; Be 100ng/ μ L with the DNA concentration dilution at last, and be kept at-20 ℃ subsequent use.
2, design 5 ' anchor primer and pcr amplification
Design contains the primer that annexs base and repeat base; Wherein annex base portion and have the grappling effect; Prevent primer and template combine slide, repeating base portion is that 2 bases repeat for 6 times, 3 or 4 bases repeat for 4 times, is used for combining with the repetition base of genomic dna; The primer that adopts has PCT6:5 '-KKVRVRV (CT)
6-3 ', PGT6:5 '-KKRVRVR (GT)
4-3 ', PCAC4:5 '-KKBDDBD (CAC)
4-3 ', synthetic by biotech firm.
Pcr amplification is that each 5 ' anchor primer is that template is carried out pcr amplification with the Scylla paramamosain genomic dna, and reaction system is 25 μ L, comprises that genomic dna template 1 μ L, 5 ' anchor primer final concentration are 0.8 μ mol/L, Mg
2+Final concentration is that 1.5mmol/L, dNTP final concentration are 0.2mmol/L, Taq archaeal dna polymerase 0.75U, 1 * PCR buffer, and replenishing sterilization distilled water to TV at last is 25 μ L; Response procedures is: 94 ℃ of preparatory sex change 5 minutes, and the annealing temperature annealing of 94 ℃ of sex change 30 seconds, primer specific was extended 30 circulations 50 seconds for 50 seconds, 72 ℃; Extended 7 minutes in 72 ℃ at last; Agarose gel electrophoresis with 1.5% detects preparatory amplified production, and reclaims the amplified production of size between 250-800bp.
3, the T carrier connects
The pcr amplification product of above-mentioned recovery is connected in the T carrier, and linked system is 10 μ L, comprises solution I 5.0 μ L, pMD19-T carrier (TaKaRa) 1.0 μ L and pcr amplification product 4.0 μ L, and 4 ℃ are spent the night.
4, clone, order-checking
To connect product and be transformed among the competent escherichia coli cell DH5 α, use carrier universal primer (M
13-47:5 '-CGCCAGGGTTTTCCCAGTCACGAC-3 '; M
13-48:5 '-AGCGGATAACAATTTCACACAGGA-3 ') clone being carried out PCR identifies.The PCR reaction system is 25 μ L, comprises that bacterium liquid 1 μ L, upstream and downstream primer final concentration are 0.4 μ mol/L, Mg
2+Final concentration is that 1.5mmol/L, dNTP final concentration are 0.2mmol/L, Taq archaeal dna polymerase 1U, 1 * PCR buffer, and replenishing sterilization distilled water to TV at last is 25 μ L; The PCR response procedures is: 94 ℃ of preparatory sex change 5 minutes, and 30 seconds, 55 ℃ annealing of 94 ℃ of sex change were extended 25 circulations 50 seconds for 50 seconds, 72 ℃; Extended 7 minutes in 72 ℃ at last.At last, select and insert the positive colony of fragment length between 250-1000bp, check order with the ABI3730 sequenator.
5, micro-satellite primers design
At first use software SSRHUNTER 1.3 to search and contain little satellite core repeating sequences, the standard of searching is: 2-5 base repeating unit, multiplicity >=4 time.The microsatellite sequence that utilizes 5.0 couples of biological software Primer Premier to contain complete flanking sequence then carries out design of primers.Primer should meet following standard: (1) primer length is between 18-25bp; (2) GC content is between 40-60%; (3) annealing temperature is between 48-60 ℃; (4) the expection length of PCR product is between 130-350bp.Primer information sees table 1 for details.
6, the pcr amplification of microsatellite marker
Utilize above-mentioned micro-satellite primers that Scylla paramamosain colony is carried out pcr amplification, its reaction system is 25 μ L, comprises that genomic dna template 1 μ L, upstream and downstream primer final concentration are 0.4 μ mol/L, Mg
2+Final concentration is that 1.5mmol/L, dNTP final concentration are 0.2mmol/L, Taq archaeal dna polymerase 1U, 1 * PCR buffer, and replenishing sterilization distilled water to TV at last is 25 μ L.The PCR response procedures is: 94 ℃ of preparatory sex change 5 minutes, and annealing temperature (table 1) annealing of 94 ℃ of sex change 30 seconds, primer specific was extended 35 circulations 50 seconds for 50 seconds, 72 ℃; In 72 ℃ of extensions 7 minutes, 4 ℃ of preservations were subsequent use at last.
7, the electrophoresis detection of PCR product
The denaturing agent (98.0% methane amide, 10mmol/L EDTA, 0.25% tetrabromophenol sulfonphthalein, 0.25% YLENE cyanogen) that in the PCR product, adds about 1/2 volume, in 95 ℃ of sex change 5 minutes, cooling fast; Go up the about 3 μ l of appearance then and in 6% denaturing polyacrylamide gel, carry out electrophoresis.Molecular weight standard is pBR322/MspI, and electrophoresis liquid is 1 * TBE, constant voltage 35-40V/cm, the about 1-1.5 of electrophoresis hour.
Dye after electrophoresis is accomplished and develop the color, its operating process is: at first with fixing 10 minutes of 70% ethanol, distillation was washed 5 minutes; Use 1.5 ‰ cma stainings 10 minutes then, distillation washed for 8 seconds; It is clear to banding pattern to develop the color with colour developing liquid (formaldehyde of 2% NaOH+4 ‰) at last, with zero(ppm) water gel is rinsed well again, has just obtained the polymorphum collection of illustrative plates of Scylla paramamosain heritable variation.
18 microsatellite markers of the Scylla paramamosain that table 1 the present invention is screened
Claims (1)
1. the screening method of a microsatellite molecular marker of scylla paramamosain comprises:
(1) extraction of Scylla paramamosain genomic dna and diluted for use;
(2) design 5 ' anchor primer and pcr amplification;
(3) clone of amplified production and order-checking;
(4) sequential analysis and little satellite special primer design;
(5) use primer that individual genomic dna in different geographical populations of Scylla paramamosain or the colony is carried out pcr amplification;
(6) denaturing polyacrylamide gel electrophoresis of use 6% detects pcr amplification product;
(7) confirm the genotype that each is individual according to the different migration distances of amplified production, obtain the microsatellite marker of 18 polymorphums altogether, thereby obtain the polymorphum collection of illustrative plates of Scylla paramamosain heritable variation;
The extraction of the Scylla paramamosain genomic dna in the said step (1) may further comprise the steps: clip Scylla paramamosain muscle tissue 100-150mg; Place 500 μ L tissue extracts to shred; The adding final concentration is that Proteinase K and the final concentration of 20mg/mL is the RNaseA of 100 μ g/mL, and 55 ℃ digested 2-3 hour; Use the phenol of volume ratio: chloroform: twice of primary isoamyl alcohol mixed solution extracting as 25:24:1; Use the absolute ethyl alcohol deposit D NA of 2 times of volumes then, after 70% washing with alcohol and seasoning, be dissolved among the TE; Be 100ng/ μ L with the DNA concentration dilution at last, and be kept at-20 ℃ subsequent use;
Design 5 ' anchor primer in the said step (2) may further comprise the steps: design contains the primer that annexs base and repeat base; Wherein annex base portion and have the grappling effect; Prevent primer and template combine slide; Repeating base portion is that 2 bases repeat for 6 times, 3 or 4 bases repeat for 4 times, is used for combining with the repetition base of genomic dna; The primer that adopts has PCT6:5 '-KKVRVRV (CT)
6-3 ', PGT6:5 '-KKRVRVR (GT)
4-3 ', PCAC4:5 '-KKBDDBD (CAC)
4-3 ';
Described pcr amplification may further comprise the steps: each 5 ' anchor primer is that template is carried out pcr amplification with the Scylla paramamosain genomic dna, and reaction system is 25 μ L, comprises that genomic dna template 1 μ L, 5 ' anchor primer final concentration are 0.8 μ mol/L, Mg
2+Final concentration is that 1.5mmol/L, dNTP final concentration are 0.2mmol/L, Taq archaeal dna polymerase 0.75U, 1 * PCR buffer, and replenishing sterilization distilled water to TV at last is 25 μ L; Response procedures is: 94 ℃ of preparatory sex change 5 minutes, and the annealing temperature annealing of 94 ℃ of sex change 30 seconds, primer specific was extended 30 circulations 50 seconds for 50 seconds, 72 ℃; Extended 7 minutes in 72 ℃ at last; Agarose gel electrophoresis with 1.5% detects preparatory amplified production, and reclaims the amplified production of size between 250-800bp;
Clone and order-checking in the said step (3) may further comprise the steps: at first be that the PCR product that reclaims is connected among the pMD19-T carrier TaKaRa, be transformed into then among the competent escherichia coli cell DH5 α, be inoculated into LB Amp
+Grow in the substratum, utilize carrier universal primer and pcr amplification method to identify positive colony again, picking inserts clip size and utilizes the ABI3730 automatic DNA sequencer DNA to check order at the positive colony of 250-1000bp;
Sequential analysis in the said step (4) and the design of little satellite special primer may further comprise the steps: at first utilize 4.0 pairs of all sequences that obtained of biological software DNAMAN to compare, remove identical sequence; Utilize biological software SSRHUNTER 1.3 to search then and contain little satellite core repeating sequences; Utilize these microsatellite sequences of BLAST method search in the GenBank DB, whether to have identical sequence again, and remove those identical sequences; The microsatellite sequence that utilizes 5.0 couples of biological software Primer Premier to contain complete flanking region at last carries out design of primers;
Wherein, described microsatellite molecular marker of scylla paramamosain and corresponding special primer sequence thereof, as follows:
Scypa01
1?tccctaccta?ccattacacc?ccattaccca?ttccccattg?cccttctgtt?catcttacct
61?gccctaatct?gtaacctgtc?actttttttc?gttgtctcgt?tgctcctcct?cctcttcctc
121?ctcctgtact?attccatctc?catcaaatag?ctacaatatt?tattcattta?gattatttac
181?ttcccaaaca?gccatatcag?ggactacaga?tctgttgctg?tctggctgtc?ctttgtaata
The special primer sequence of Scypa01 mark:
F:TCCCTACCTACCATTACACCC
R:TATTACAAAGGACAGCCAGACA
Scypa02
The special primer sequence of Scypa02 mark:
F:TCTGTAATCAGACCAAGGAGGT
R:CAAAATAGCCATACTGGAAGC
Scypa03
The special primer sequence of Scypa03 mark:
F:GCGGTTCATTTGCTTCG
R:CGAGACTGGGTTGTCCTTA
Scypa04
The special primer sequence of Scypa04 mark:
F：CCACTCCTGCCATCCTCATT
R:CCCAGCGGCATCTTTGTC
Scypa05
1?ggatagttgc?tggttgatga?agcctggaat?tgtggtagtc?atcagaatag?tagtagtagt
61?agtagttgta?gtagttgcaa?ttttctacta?ctaatactaa?tactactata?actactacta
121?ttactattac?tatcaccact?atttacctgc?atgcaaagcc?gcttcaatta?acattctacc
181?acaattcgcc?gcagacccc
The special primer sequence of Scypa05 mark:
F:GGATAGTTGCTGGTTGATGAAG
R:GGGGTCTGCGGCGAAT
Scypa06
The special primer sequence of Scypa06 mark:
F:TGAGAGAGGCAGGGTAGGAA
R:AAAGCGTTAACTTCCATTACACA
Scypa07
1?gcgctctgag?gcaagaagag?aaactaacca?gtaatagtag?aagagcacac?acacacacac
61?acacacacac?acacacacat?actcataggg?gaagggcagc?gagtcaaggc?ttcacgtgct
121?cgaatgaaag?ctttggtact?caccaggatc?ttgtttcatc?acctcttttc?ttatttcttc
181?ctttttcacc?cttttttttt?tgtaggagga?aagggtgatg?ggagatgtat?ggctaagc
The special primer sequence of Scypa07 mark:
F:GCGCTCTGAGGCAAGAAG
R:GCTTAGCCATACATCTCCCAT
Scypa08
1?acacgagaca?gagggaggct?cctgtactac?acatcactca?gggaagagag?agaagtaaat
61?ctttggggta?aaaacagtac?cagaaactcc?gtaacttgac?tgtaactttg?agctcataaa
121?gttttcaagg?aagttgtact?ctctctctct?ctctctctag?atggattaga?acacagaatc
181?ttgtatctcg?aacccgg
The special primer sequence of Scypa08 mark:
F:ACACGAGACAGAGGGAGGC
R:CCGGGTTCGAGATACAAGAT
Scypa09
1?cgagactcag?atcggatgat?ggtgatgaag?cacgaggaat?gcatgttgat?ctggggcgtg
61?gagggctgtg?atggtggtgg?tggtgatggt?ggtgggagta?gtagtagtag?tagtagtaat
121?agttgaggtt?attgttgttg?cagtcgtggt?agtagtagta?gtagtagtaa?tagtagtagt
181?aacagcagta?acaattgtaa?tcactgctag?tagcgg
The special primer sequence of Scypa09 mark:
F:CGAGACTCAGATCGGATGATG
R:CCGCTACTAGCAGTGATTACAAT
Scypa10
1?tagttctttc?ttccttctac?cgataccacc?accaccacca?ccacccccaa?aaccacaaga
61?gcaccacacc?ttaaaatacc?agcagcagta?acattagtag?ctctgttagc?actgatagca
121?aagacaatgg?agggaaaaaa?aaaaaaaaca?gaataagaaa?cttgtcaagg?caagaggtga
181?ggtggtcagc?caggcaggtg
The special primer sequence of Scypa10 mark:
F:TAGTTCTTTCTTCCTTCTACCGAT
R:CACCTGCCTGGCTGACC
Scypa11
1?aacgctacat?catactgcgt?acttcccagt?ggtgcacaca?cacacacaca?cacacacaca
61?cacacacacc?ataagtggat?ggataagtga?gtatctatgg?attacttatg?aaaatgagag
121?cataaagtat?tcttgttttt?gtagtggtgg?tggtgaagta?tttatagaag?tagtagtcgt
181?agtagtagta?gtaatatagt?agtagaagta?agcagaaata?gcaacag
The special primer sequence of Scypa11 mark:
F:AACGCTACATCATACTGC
R:CTGTTGCTATTTCTGCTT
Scypa12
1?accgccattc?attccattca?cccaccgttt?tgaatttaca?agtttattat?gaagaaaacg
61?caaagtacag?aagtcacgga?ggacagacag?acagacagac?agacagacag?actttacacg
121?caggcagggt?aactgaagga?ggccagacgc?aggtatagtg?gggctcggct?gttccttta
The special primer sequence of Scypa12 mark:
F:ACCGCCATTCATTCCATT
R:TAAAGGAACAGCCGAGCC
Scypa13
The special primer sequence of Scypa13 mark:
F:AGCGTCTGTCCACCCTTAG
R:GACTTTCCCACAACCTCGTAT
Scypa14
1?gacccgattt?gcatctttag?tttttttttt?ttttttatgc?ataaagaatg?ccttccaaaa
61?caacaacaac?aacaacaaca?acaaaagtgg?gataaacaaa?aagcccacta?gtttgcctct
121?tcccagaatt?aaaaaaaaaa?aaaaaaacgg?gacaagatca?ccgaaacgga?agcc
The special primer sequence of Scypa14 mark:
F:CCCGATTTGCATCTTTAGTT
R:CTTCCGTTTCGGTGAT
Scypa15
1?ccgcgaaaca?aggagaacat?ttaagggttc?tttcttagta?cagggaataa?agcaggtgtg
61?gtgatgggtg?tgtggtgcag?cggttgttgt?ggtgtatgtt?agtggtggtg?tggtggtgtg
121?tttgcgtggt?gcatgaggga?gtgctttgtg?tgtgtgtgtg?tgtgtgtgtg?tgtgtgtgtg
181?tgtgtgtgtg?tgtgtgtata?gtttgatgga?tgctacaccg
The special primer sequence of Scypa15 mark:
F:GCGAAACAAGGAGAAC
R:GTGTAGCATCCATCAAAC
Scypa16
1?ctgcctcttt?aatgttgact?cctcactatt?ttatttattt?ttgttttgtt?ttaccagtga
61?attttagaat?gtctacccat?ttctttttgt?tgtattgatt?gttgttgttg?ttgttgttgt
121?tgttgttgct?tctgctacta?ctactaatac?taataccact?acatcttttg?tggtag
The special primer sequence of Scypa16 mark:
F:CTGCCTCTTTAATGTTGACTCC
R:CTACCACAAAAGATGTAGTGGTATT
Scypa17
1?taactggttg?gtcggctgat?tgaaagtgct?ggagaaaacg?gaataatttt?acgccgcagg
61?acgaaaagaa?gtgattggat?caggaatctg?aatcgaagag?gaaagatact?actgtagcta
121?agagagagag?agagagagag?agagagagag?agagagattt?ttttttccct?cagtatacac
181?tctaatggaa?aaataataac?agtactaaca?cacagaactc?agcttacgga?cgactc
The special primer sequence of Scypa17 mark:
F:ACTGGTTGGTCGGCTGATTG
R:GTCGTCCGTAAGCTGAGTTC
Scypa18
1?accaaaaaca?agaaatatca?ccactatcat?cattattcct?accacaacaa?caacaacaac
61?aacaaaacca?cgcccacagg?aaccagccac?caaaacctcc?ccaaaaaaac?acccaccaaa
121?atatattaaa?accgatacac?atatatatac?gtatacatcc?cctgggcagc?ctcacatatt
181?tttcctcttc?ccttccgttt?cctcccagag?cgacaaattt?tggtggcgaa?ggagacggta
The special primer sequence of Scypa18 mark:
F:CAAAAACAAGAAATATCACCA
R:CCGTCTCCTTCGCCA；
Use primer in the said step (5) is 25 μ L to the reaction system that individual genomic dna in different geographical populations of Scylla paramamosain or the colony carries out pcr amplification, comprises that genomic dna template 1 μ L, upstream and downstream primer final concentration are 0.4 μ mol/L, Mg
2+Final concentration is that 1.5mmol/L, dNTP final concentration are 0.2mmol/L, Taq archaeal dna polymerase 0.75U, 1 * PCRbuffer, and replenishing sterilization distilled water to TV at last is 25 μ L; Response procedures is: 94 ℃ of preparatory sex change 5 minutes, and the annealing temperature annealing of 94 ℃ of sex change 30 seconds, primer specific was extended 35 circulations 50 seconds for 50 seconds, 72 ℃; Extended 7 minutes in 72 ℃ at last;
The denaturing polyacrylamide gel electrophoresis of the use 6% in the said step (6) detects pcr amplification product: after 95 ℃ of sex change of PCR product, the about 3 μ L of last appearance carry out electrophoresis in 6% polyacrylamide gel; Obtained the PCR product electrophoretic image of Scylla paramamosain after dyed, the colour developing.
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| MA H Y，MA C Y，MA L B，et al．.Novel polymorphic microsatellite makers in Scylla paramamosain and cross-species amplification in related crab species.《Journal of Crustacean Biology》.2010,第30卷(第3期),441－444. * |
| XU X J,WANG G Z,WANG K J,et al.Isolation and characterization of ten new polymorphic microsatellite loci in the mud crab,Scylla paramamosain..《Conservation Genetics》.2009,第10卷(第6期),1877-1878. * |
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