CN103397030B - Hemifusus tuba micro-satellite site and primer - Google Patents
Hemifusus tuba micro-satellite site and primer Download PDFInfo
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Abstract
The invention aims at providing a hemifusus tuba micro-satellite site and a pleomorphic primer, namely providing the micro-satellite site with six hemifusus tubas, wherein the nucleotide sequence of the micro-satellite site is any one of SEQ ID NO:1-6. The invention further provides a primer which is designed from the micro-satellite site, wherein the primer is used for detecting group genetic diversity of the hemifusus tubas. According to the invention, 6 micro-satellite sites are screened from a hemifusus tubas public database, and specific primers are designed according to flanking sequences at the two ends of the micro-satellite, so that the amplification result has high polymorphism and stability, and can be used for the group genetic diversity detection and the genetic relationship identification of the hemifusus tuba, the construction of a genetic linkage map as well as the molecular marker-assisted selection field.
Description
Technical field
The invention belongs to molecular biology DNA marker technical field, be specifically related to a kind of Trumpet shell whelk microsatellite locus and primer.
Background technology
Microsatellite DNA (Microsatellite): be also called STR (short tandem repeats, or simple sequence repeats (simple sequence repeats STRs), SSRs), refer to that by 1-6 Nucleotide be the DNA repeating unit of simply connecting that core sequence forms, tumor-necrosis factor glycoproteins from beginning to end.In all biological species studied so far, all found its existence, and distribution density is very large, shows the polymorphism of height.Because micro-satellite has rich polymorphism, follows the feature such as Mendel's law of segregation, codominant inheritance, become the most widely used genetic marker in population genetics, pedigree analysis, linkage map structure, Study on Evolution.
Trumpet shell whelk (Hemifusus tuba) is commonly called as and rings spiral shell, angle spiral shell, be under the jurisdiction of Mollusca (Mollusca), Gastropoda (Gastropoda), Caenogastropoda (Neogastropoda), Kui Luo section (Melongenidae), live in rice sand shale seabed more than deep water offshore 10-40, adult shell height 12-20cm (about 150-300g), more than maximum shell height 30cm (body weight 500g), be the economic snails that shallow sea is relatively large.Distribution is had the coastal of each province such as China Zhejiang, Jiangsu, Guangxi and Guangdong.In recent years along with the increase of market demand, cause the overfishing of wild Trumpet shell whelk, add the artificial interference factors such as the pollution of ocean environment, coastal Trumpet shell whelk natural resources amount is reduced day by day.In order to realize the Sustainable Development and Utilization of Trumpet shell whelk resource, in the urgent need to investigating the Present Situation of Natural Resources of its population.Microsatellite molecular marker is widely used in the population genetic study field of species, is a kind of efficient, reliable molecule marker, therefore, filters out effective microsatellite marker and can provide power instrument for the population genetic Epidemiological Analysis of Trumpet shell whelk resource.
Summary of the invention
The object of this invention is to provide Trumpet shell whelk microsatellite locus and polymorphism primer, namely the microsatellite locus of 6 Trumpet shell whelks is provided, and corresponding polymorphism primer, for the population genetics of Trumpet shell whelk, Parentage determination and molecular mark technology provide effective instrument.
One aspect of the invention provides Trumpet shell whelk microsatellite locus, and its nucleotide sequence is any one in SEQ ID NO:1-6.
The present invention also provides the primer pair designed from above-mentioned 6 microsatellite locus, and wherein sequence is the primer pair of the microsatellite locus design of SEQ ID NO:1, and its upstream and downstream sequence is respectively SEQ ID NO:7, SEQ ID NO:8.
Sequence is the primer pair of the microsatellite locus design of SEQ ID NO:2, and its upstream and downstream sequence is respectively SEQ ID NO:9, SEQ ID NO:10.
Sequence is the primer pair of the microsatellite locus design of SEQ ID NO:3, and its upstream and downstream sequence is respectively SEQ ID NO:11, SEQ ID NO:12.
Sequence is the primer pair of the microsatellite locus design of SEQ ID NO:4, and its upstream and downstream sequence is respectively SEQ ID NO:13, SEQ ID NO:14.
Sequence is the primer pair of the microsatellite locus design of SEQ ID NO:5, and its upstream and downstream sequence is respectively SEQ ID NO:15, SEQ ID NO:16.
Sequence is the primer pair of the microsatellite locus design of SEQ ID NO:6, and its upstream and downstream sequence is respectively SEQ ID NO:17, SEQ ID NO:18.
Microsatellite polymorphism primer of the present invention is used for the multifarious detection of Trumpet shell whelk population genetic, and step is as follows:
1) extraction of genomic dna: the genomic dna adopting phenol chloroform method extracting Trumpet shell whelk abdominal foot muscle tissue;
2) microsatellite PCR amplification: with Trumpet shell whelk genomic dna for template, adopts the primer of design to carry out PCR reaction, obtains the individual micro-satellite amplified production of Trumpet shell whelk;
3) electrophoresis detection amplified production: adopt denaturing polyacrylamide gel electrophoresis and argentation to detect micro-satellite amplified production;
4) analysis of genetic diversity: according to the molecular size range determination genotype of the micro-satellite amplified production of each individuality, adopts GENEPOP4.0.10 to calculate genetic diversity parameter.
The present invention screens 6 microsatellite locus from Trumpet shell whelk transcript profile cDNA, and increase at the flanking region design Auele Specific Primer at micro-satellite repetitive sequence two ends, the product obtained has polymorphism and the stability of height, can be used for the fields such as the population genetics of Trumpet shell whelk, Genetic relationship, molecular mark.
Accompanying drawing explanation
Fig. 1: the qualification success ratio of 6 microsatellite locus in the Trumpet shell whelk family of different parent's quantity.
Embodiment
Below in conjunction with specific embodiment, method of the present invention is described in detail, for the experimental technique of actual conditions unreceipted in embodiment, usually can condition routinely, condition as described in " Molecular Cloning: A Laboratory guide " that J. Pehanorm Brooker (Sambrook) etc. is write, or run according to the condition that manufacturer advises.
One, the screening of microsatellite locus
1, the sequence containing microsatellite locus is searched:
Searching of microsatellite sequence is carried out with software SSRHUNTER1.3 (http://www.biosoft.net/dna/SSRHunter.htm) from the sequence in the complete individual transcript profile library of Trumpet shell whelk built; Optimum configurations is search the sequence containing two bases, three bases and four base repeat numbers more than 5 times.Filter out 20 sites of repeating containing micro-satellite altogether, and therefrom design the detection that primer carries out polymorphism.
2, micro-satellite primers design:
From the gene order containing micro-satellite, choose the sequence meeting design of primers and use PrimerPremier5.0 to carry out design of primers.Significant parameter is set to: primer length 18-25p, PCR primer fragment length scope 100-350bp, the suitableeest annealing temperature 55-65 DEG C.GC content, generally between 40%-60%, avoids secondary structure to occur as far as possible.
3, the primer of design is carried out polymorphic detection
1) extraction of genomic dna:
Adopt the genomic dna of phenol chloroform method extracting 30 Trumpet shell whelk abdominal foot muscle tissues;
2) amplification of microsatellite PCR:
With Trumpet shell whelk genomic dna for template, the micro-satellite primers sequence of design is adopted to carry out pcr amplification; Reaction system 10 μ L, comprises 2 × Power Taq PCR Master Mix (BioTeke, Beijing, China) 5 μ L, and each 1 μM of forward and reverse primer, genomic dna is about 100ng.PCR response procedures is 94 DEG C of denaturation 3min, then carries out 35 amplification cycles, and each circulation comprises 94 DEG C of sex change 1min, annealing temperature 1min (each primer annealing temperature is in table 2), and 72 DEG C extend 1min, and last 72 DEG C extend 5min.
3) amplified production electrophoresis:
Adopt 8% denaturing polyacrylamide gel electrophoresis to detect amplified production, 800W invariable power electrophoresis is separated for 1-1.5 hour.Offset plate passes through the glacial acetic acid solution 30min of 10%, distilled water 6min, 0.2% silver nitrate solution 30min, the sodium carbonate of 3% develops the color 30 seconds, namely termination reaction obtains amplified production fragment, adopts 10-bp DNA ladder (Invitrogen Inc.) to detect allelotrope position as marker.
4) analysis of genetic diversity:
According to the molecular size range determination genotype of every individual micro-satellite amplified production, adopt GENEPOP4.0.10 to calculate genetic diversity parameter, thus filter out there is the micro-satellite primers of polymorphism and the site of correspondence.
Detect through diversity, the present invention filters out the microsatellite locus that 6 have genetic polymorphism altogether, and its nucleotide sequence is respectively SEQ ID NO:1-6, and the information of the polymorphism primer of its design is as shown in table 1.
Table 1: 6 microsatellite locus of the present invention and corresponding primer information
Increase with the genomic dna of primer pair of the present invention 30 Trumpet shell whelk muscle tissues, analysis of genetic diversity result shows the allelotrope number of each microsatellite locus from 7-10 not etc., and average allelotrope number is 7.7.Observation heterozygosity (H
o) scope from 0.633 to 0.929, expect heterozygosity (H
e) scope from 0.724 to 0.899(table 2).Prove that the microsatellite locus of the present invention's screening and the primer of design have genetic polymorphism.
The relevant information of the annealing temperature, fragment length, polymorphism etc. of table 2:6 microsatellite locus
Wherein N
arepresent allelotrope number, H
orepresentative observation heterozygosity, H
eheterozygosity is expected in representative
5) Parentage determination analysis:
With the gene frequency information of 6 microsatellite locus in 30 Trumpet shell whelk individualities for foundation, these 6 microsatellite locus of CERVUS3.0 software estimation are utilized to be respectively 5 at parent's logarithm, 10,15,20,25, the success ratio of Parentage determination under the condition of 30.Detect parameters arranges and the following is: analog submodule algebraically order 10000, parent's verification and measurement ratio 100%, site verification and measurement ratio 100%, somatotype specific inaccuracy 1%, and confidence level is 95%, and parent's sex is unknown.Detected result as shown in Figure 1.
Qualification result shows: under 95% degree of confidence, and 6 microsatellite locus Parentage determination success ratio when parent number is 30 pairs is 89%; When parent number is less than or equal to 20 pairs, qualification success ratio reaches more than 96%.As can be seen here, microsatellite marker of the present invention has higher determination rates, and it is feasible for utilizing 6 microsatellite locus of the present invention in Trumpet shell whelk colony, carry out paternity test.
Micro-satellite primers of the present invention also can be used for the area researches such as the population genetic variations of Trumpet shell whelk, recruitment evaluation of releasing, Genetic relationship and molecular mark.
Claims (2)
1. a micro-satellite primers pair, is characterized in that, described micro-satellite primers is to being that the Trumpet shell whelk microsatellite locus being SEQ ID NO:1 from nucleotide sequence designs, and its upstream and downstream sequence is respectively SEQ ID NO:7, SEQ ID NO:8.
2. micro-satellite primers according to claim 1 is to for the multifarious detection of Trumpet shell whelk population genetic, comprises the steps:
1) extraction of genomic dna: the genomic dna adopting phenol chloroform method extracting Trumpet shell whelk muscle tissue;
2) microsatellite PCR amplification: the primer adopting design, Trumpet shell whelk genomic dna is that template carries out PCR, obtains the individual micro-satellite amplified production of Trumpet shell whelk;
3) electrophoresis detection amplified production: adopt denaturing polyacrylamide gel electrophoresis and argentation to detect micro-satellite amplified production;
4) analysis of genetic diversity: according to the molecular size range determination genotype of the micro-satellite amplified production of each individuality, adopts GENEPOP 4.0.10 to calculate genetic diversity parameter.
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| CN104450915A (en) * | 2014-12-09 | 2015-03-25 | 广西大学 | Hemifusus tuba microsatellite loci, and primers and application of hemifusus tuba microsatellite loci |
| CN107574167B (en) * | 2015-12-15 | 2020-09-22 | 浙江师范大学 | Hyriopsis cumingii microsatellite marker and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103060317A (en) * | 2012-12-29 | 2013-04-24 | 宁波大学 | Sepiella maindroni microsatellite loci and primers |
| CN103114088A (en) * | 2013-03-06 | 2013-05-22 | 宁波大学 | Sepiella maindroni microsatellite sites and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103060317A (en) * | 2012-12-29 | 2013-04-24 | 宁波大学 | Sepiella maindroni microsatellite loci and primers |
| CN103114088A (en) * | 2013-03-06 | 2013-05-22 | 宁波大学 | Sepiella maindroni microsatellite sites and application thereof |
Non-Patent Citations (2)
| Title |
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| 应用微卫星DNA 标记探讨我国长江中下游地区湖北钉螺群体遗传结构;李石柱 等;《中国寄生虫学与寄生虫病杂志》;20120830;第30卷(第4期);"材料与方法"部分 * |
| 细角螺微卫星DNA富集文库构建及特征分析;李清荟 等;《海洋科学》;20130415;第37卷(第4期);"材料和方法"、"结果"部分 * |
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