CN102146437B - Detecting method for Chinese prawn ES11 microsatellite marker - Google Patents
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- 241000238557 Decapoda Species 0.000 title claims abstract description 59
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Abstract
The invention relates to a detecting method for a Chinese prawn ES11 microsatellite marker, which comprises the following steps of: firstly, extracting a Chinese prawn genome DNA; designing specific primers at both ends of a sequence by utilizing a ES11 microsatellite-contained core sequence in a Chinese prawn cDNA library; carrying out PCR (Polymerase Chain Reaction) amplification on the genome DNA of different geographical groups of Chinese prawns or individuals in the Chinese prawn group; carrying out denatured polyacrylamide gel electrophoresis separation on an obtained PCR product to obtain a polymorphism map for presenting high hereditary variation of the Chinese prawns in the ES11 core sequence area; and analyzing a stripe of the map to obtain the genotype of each individual of the Chinese prawn in the ES11 locus. The invention can rapidly obtain the hereditary variation map of Chinese prawn ES11 gene locus, and the method is simple and convenient and can visually detect the genotype of each individual of the Chinese prawn; and the invention is mainly applied to Chinese prawn group genetic marker, genealogy authentication, construction of the genetic map, and the like.
Description
Technical field:
The invention belongs to Chinese prawn dna molecular genetic marker technology, is the microsatellite marker detection method of a kind of Chinese prawn genetic polymorphism in the ES11 site.
Background technology:
Before the present invention makes, similar research report is arranged, (aquatic product journal, 2001a such as the domestic Xu Peng of the Institute of Oceanology of the Chinese Academy of Sciences both at home and abroad; Oceanologia et Limnologia Sinica, 2001b; Aquatic product journal; 2003) " recombinant clone that contains little satellite with PCR method rapid screening Chinese prawn " delivered, " screening of Chinese prawn microsatellite DNA " and papers such as " researchs of screening microsatellite marker from Chinese prawn ESTs "; Once reported Chinese prawn portion gene group library structure, contain the contents such as screening of microsatellite sequence; And little satellite core sequence is estimated according to the standard that Weber (U.S., the assorted Field biomedical research foundation of horse, 1990) proposes; But his these results of study only limit to study the acquisition of microsatellite sequence, do not have further application and development.Liu Ping etc. (Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science, Oceanologia et Limnologia Sinica, 2004) have developed 8 SSR sites, and have accomplished the analysis of genetic diversity of 3 natural populations of Chinese prawn.Sun Zhao rather waits (Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science, Chinese aquatic science, 2005) with 6 microsatellite markers 5 familys of Chinese prawn to be carried out pedigree discriminating and genetic diversity research.Dong Shirui etc. (Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science, ocean journal, 2006) have identified the sibship of 6 familys that the stud mating of 2 familys of Chinese prawn mixed breed is bred with 2 pairs of little satellites.Meng Xianhong etc. (Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science, marine fishery research, 2006) screen 11 pairs of primers and have polymorphum from 33 pairs of micro-satellite primers.Institut Francais de Recherche Pour L'Exploitation de la Mer-ifremer (IFREMER) found 10 microsatellite markers in 1999 from blue prawn population, from tigar prawn, find 3 microsatellite markers, had brought into play effect when these are marked at the different family that identification raises together with.The Chinese prawn microsatellite marker of registering among the GenBank at present has 110; Still extremely lack but can be used for the quantity that genetic linkage maps makes up with the microsatellite marker of pedigree identification, do not see the report of aspect application such as structure that Chinese prawn microsatellite polymorphism collection of illustrative plates is arranged, specificity genetic marker both at home and abroad as yet.
Summary of the invention:
The present invention its objective is and proposes a kind of Chinese prawn dna molecular genetic marker detection method; The main little satellite core sequence that contains that utilizes in the Chinese prawn cDNA library of setting up; Carrying out PCR in its design specific primers at both ends detects; Thereby detect of the heritable variation of each individuality of Chinese prawn apace, obtain this primer, detect each individual genotype intuitively through collection of illustrative plates to Chinese prawn polymorphum collection of illustrative plates in this little satellite district.
The present invention accomplishes according to following operational aspect:
At first extract Chinese prawn genomic dna and diluted for use; Utilize again and contain the little satellite core sequence of ES11 in the Chinese prawn cDNA library, in its sequence design specific primers at both ends; Use this primer that individual genomic dna in different proles of Chinese prawn or the Chinese prawn crowd is carried out pcr amplification then, the PCR product is detected; The band that utilizes product to occur is analyzed, and confirms the genotype that each is individual, and obtains the genetic polymorphism collection of illustrative plates of Chinese prawn.
Extract the Chinese prawn genomic dna, its dilution is 50ng/ μ l, add 2 μ l in each PCR reaction, the reaction TV is 20 μ l.The specific primer sequence of the little satellite core sequence of ES11 is respectively: normal chain 5 '-GGT GTT TCG GTC TGG TAT T-3 ', and minus strand 5 '-GCT GGG AGT GAC TAG GGA T-3 ', the annealing temperature when using this primer is 50 ℃.
The application of sample parameter of pcr amplification is: each PCR reaction TV is 20 μ l, comprises 100ng Chinese prawn genomic dna; 10X PCR Buffer, 2.0 μ l; Mg
2+2.0mmol/L; Taq enzyme 1U; Each 0.1mmol/L of dNTP; Each 0.2 μ mol/L of primer of the present invention; Add ddH
2O to 20 μ l.The programparameter that the PCR appearance is set when using this primer is: 94 ℃ of sex change 5min; 94 ℃ of 40sec, 64 ℃ of 40sec, 72 ℃ of 40sec, 35 circulations; 72 ℃ are extended 10min, 4 ℃ of preservations.
The detection of PCR product: at 8% denaturing polyacrylamide gel, the permanent power electrophoresis of 12W separated in 1-1.5 hour with the PCR product.With the sodium carbonate solution colour developing of offset plate through silver nitrate solution 20min → 3% of glacial acetic acid solution 20min → zero(ppm) water 6min → 0.1% of 10% several minutes, termination reaction can obtain the polymorphum collection of illustrative plates of Chinese prawn at the ES11 locus.
Technology of the present invention has following characteristics:
(1) the present invention can obtain the heritable variation collection of illustrative plates of the ES11 genetic marker locus of Chinese prawn efficiently, and method is easy, and the gained result can detect each individual genotype of Chinese prawn intuitively, thereby distinguishes homozygote and heterozygote individuality;
(2) the present invention is mainly used in genetic marker, genealogical identification and construction of genetic atlas etc. between Chinese prawn colony.The PCR primer is a core of the present invention, in the total crowd of Chinese prawn detects, presents polymorphum.
Description of drawings:
Fig. 1: ES11 primer of the present invention is to the detection collection of illustrative plates of Chinese prawn 20 individuals, and numbering 1-20 is 20 individuals of Chinese prawn, and M is DL 100 standard molecular weights.
Embodiment:
Combine accompanying drawing to be described in detail the present invention through embodiment below at the little satellite core sequence of Chinese prawn ES11 dna molecular genetic marker technological method.
At first extract Chinese prawn genomic dna and diluted for use; Utilize the little satellite core sequence that contains in the Chinese prawn cDNA library again, in its sequence design specific primers at both ends; Use this primer that individual genomic dna in different proles of Chinese prawn or the Chinese prawn crowd is carried out pcr amplification then, the PCR product is detected; The band that utilizes product to occur is analyzed, and confirms the genotype that each is individual, and obtains the genetic polymorphism collection of illustrative plates of Chinese prawn.
1, the extraction of Chinese prawn genomic dna: get prawn muscle tissue 100mg, put into the 1.5ml centrifuge tube after shredding, add TE solution (10mmol/L Tris-C1,10mmol/L EDTA) the 475 μ l of pH8.0, grind with grinding rod.Add 10%SDS solution 25 μ l, mixing.Add 20mg/ml Proteinase K 4 μ l, mixing, 55 ℃ of digestion 2.5～3h.Re-distilled phenol extracting twice, each 10min, the centrifugal 5min of 12000 commentaries on classics/min gets supernatant; Phenol: chloroform (1: 1) extracting once, 10min, the centrifugal 5min of 12000 commentaries on classics/min gets supernatant; 5min of chloroform extracting, the centrifugal 5min of 5000g gets supernatant.The 5mol/L NaCl solution that adds 1/25 volume adds-20 ℃ absolute ethyl alcohol deposit D NA 15min of two volumes again behind the mixing; The DNA that chooses, the washing with alcohol with 70% tens of minutes makes DNA dry, and is after fully dissolving with aqua sterilisa 500 μ l, quantitatively that its concentration that is diluted to 50ng/ μ l is subsequent use.
2, the design of micro-satellite primers: in Chinese prawn cDNA library, contain on the basis of the little satellite core sequence of ES11; The sequence of utilizing the microsatellite DNA both sides is in the high conservative property of same species with respect to core sequence; In its design specific primers at both ends, amplify little satellite fragment in this site in view of the above with it.Because little satellite core sequence mutation rate is higher relatively, causes the increase or the minimizing of microsatellite DNA core sequence multiplicity, i.e. the variation of microsatellite DNA sequence length, this is the root that detects microsatellite polymorphism.The specific primer sequence at core sequence two ends, little satellite of the present invention district is: normal chain 5 '-GGT GTT TCGGTC TGG TAT T-3 ', and minus strand 5 '-GCT GGG AGT GAC TAG GGA T-3 ', the annealing temperature when using this primer is 50 ℃.
3, pcr amplification: application of sample at first, the application of sample amount is following: Chinese prawn genomic dna (50ng/ μ l), 2 μ l; 10X PCR Buffer, 2.0 μ l; Mg
2+(25mmol/L), 2 μ l; Taq enzyme (5U/ μ l), 0.2 μ l; DNTP (each 2.5mmol/L), 2 μ l; Primer of the present invention (each 10mmol/ μ l), 2 μ l; Add aqua sterilisa to 20 μ l.Secondly, carry out the PCR reaction, its pcr amplification appearance programparameter is: 94 ℃ of sex change 5min; 94 ℃ of 40sec, 64 ℃ of 40sec, 72 ℃ of 40sec, 35 circulations; 72 ℃ are extended 10min, 4 ℃ of preservations.
4, after the detection of PCR product: PCR reaction finishes, product is carried out electrophoresis in 8% denaturing polyacrylamide gel, the power of electrophoresis apparatus is 12 watts, and electrophoresis time can stop about 1-1.5 hour.With the sodium carbonate solution colour developing of offset plate through silver nitrate solution 20min → 3% of glacial acetic acid solution 20min → zero(ppm) water 6min → 0.1% of 10% several minutes, termination reaction promptly obtained polymorphum collection of illustrative plates as shown in Figure 1.
Claims (1)
1. the detection method of a Chinese prawn ES11 microsatellite marker is characterized in that method is: at first extract Chinese prawn genomic dna and diluted for use; Utilize again and contain the little satellite core sequence of ES11 in the Chinese prawn cDNA library, in its sequence design specific primers at both ends; Use this primer that individual genomic dna in different proles of Chinese prawn or the Chinese prawn crowd is carried out pcr amplification then, the PCR product is detected; The band that utilizes product to occur is analyzed, and confirms the genotype that each is individual, and obtains the genetic polymorphism collection of illustrative plates of Chinese prawn;
The specific primer sequence of the little satellite core sequence of described ES11 is respectively: normal chain 5 '-GGT GTT TCG GTC TGG TAT T-3 ', and minus strand 5 '-GCT GGG AGT GAC TAG GGA T-3 ', the annealing temperature when using this primer is 50 ℃;
The little satellite core sequence of said ES11 is:
GGTGTTTCGG?TCTGGTATTA?TTCCGAAATT?AGAGTTTATT?CTATATATAT
ATATATATAT?ATTGTTTGCC?AAAGACTGAG?GTGAAGGAAC?GAGATACTGG
TAAATCGCGT?GAACAGATAT?TCGCTGGAAT?ATGAATTGTT?GCATTTCCCA
GTAGAGGAAA?GAGGATATGG?TGAACAAAGA?GACAGTATTT?GTATAAAGAA
GAGACGGAAA?TCCCTAGTCA?CTCCCAGC。
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Non-Patent Citations (3)
| Title |
|---|
| 刘萍等.中国对虾微卫星DNA多态性分析.《自然科学进展》.2004,第14卷全文. * |
| 张留所等.微卫星筛选及其在对虾遗传学研究中的应用.《海洋科学》.2007,第31卷(第2期),全文. * |
| 徐鹏等.从中国对虾ESTs中筛选微卫星标记的研究.《水产学报》.2003,第27卷(第3期),全文. * |
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Application publication date: 20110810 Assignee: QINGDAO ZHUOYUE OCEAN GROUP CO.,LTD. Assignor: YELLOW SEA FISHERIES Research Institute CHINESE ACADEMY OF FISHERY SCIENCES Contract record no.: 2015370000071 Denomination of invention: Detecting method for Chinese prawn ES11 microsatellite marker Granted publication date: 20121114 License type: Exclusive License Record date: 20150430 |
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