CN103605913B - Method applied to identification of pacific oyster family - Google Patents
Method applied to identification of pacific oyster family Download PDFInfo
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- CN103605913B CN103605913B CN201310660177.XA CN201310660177A CN103605913B CN 103605913 B CN103605913 B CN 103605913B CN 201310660177 A CN201310660177 A CN 201310660177A CN 103605913 B CN103605913 B CN 103605913B
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Abstract
The invention discloses a method applied to identification of pacific oyster family and belongs to the technical field of genomic molecular markers. The method comprises the following steps: (1) searching type I and type II SNP mutation sites in an SNP database acquired by an Illumina GoldenGate method; (2) establishing genotype database files of filial generations and parents, and acquiring genetic parameters of each SNP site by utilizing Cervus 3.0 software; (3) performing family simulation identification and analysis according to the allele frequency of each site by utilizing the Cervus 3.0 software, performing actual family identification and analysis, calculating a LOD value of a candidate parental pair, and selecting parents with the highest LOD value being positive value as real parents. According to the invention, an SNP marking method applied to identification of pacific oyster family is developed, and the reaction system and polymerase chain reaction (PCR) amplification conditions are optimized. The experiments prove that the primers have the characteristics of high repeatability, high stability, high solubility curve resolution and the like, and a foundation is laid for breeding of the pacific oyster family.
Description
Technical field
The invention belongs to genome molecules labelling technique field, is related to a kind of side suitable for Pacific oyster Parentage determination
Method.
Background technology
Concha Ostreae is subordinate to Mollusca, Bivalvia, Margarita mesh, and its meat flavour is delicious, nutritious, from the ancient times just by
The mankind eat, with very high economic worth and medical value.Concha Ostreae is worldwide distribution monoid, is had now been found that more than 100
Kind, respectively border on the sea the national economic shellfish for nearly all having production, being current China and world wide production maximum in the world.
Used as the big cultivated shellfish of the first in the world, the genetic improvement of Concha Ostreae is always subject to generally weighing for domestic and international expert and scholar
Depending on, and carried out numerous studies work.Although China be oyster culture big country, but is not also oyster culture power, there is something lost
The problems such as Upgrading relatively lags behind, Study on Genetic Basis is relatively weak, breeding is deficient is passed, is had a strong impact on and to constrain China male
The healthy aquaculture of oyster and genetic improvement work.Families selecting breeding is to maintain excellent economic characters and obtains improved seeds and product
The important means of system, extensively applies in animals and plants.Completely, accurate pedigree information can avoid inbreeding, keep species
Genetic diversity, the genetic improvement work to promoting shellfish has important realistic meaning.SNP（Single nucleotide polymorphism）Refer to
Single nucleotide variation in DNA sequence, is the third generation molecular genetic marker after microsatellite marker, with genome distribution
Extensively, the advantages of being easy to detection, high hereditary stability, there is obvious advantage in Parentage determination.High-resolution fusion curve（HRM）
It is a kind of SNP typing new techniques that analysis is dissolved based on high-resolution, with high flux, low cost, easy-operating feature, extensively
The general research for being applied to the fields such as hereditism, genomics, biology.Using modern molecular labelling new technique, set up a set of suitable
For the SNP Parentage determination systems of Pacific oyster, will be to determine that sibship provides effective way between individuality, to avoid inbreeding
Decline provides strong scientific basis, is that the research work such as family selective breeding, structure inbred line, the plasm resource protection of Concha Ostreae are established
Basis.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of method suitable for Pacific oyster Parentage determination, contributes to
Quickly, accurately differentiate the Pacific oyster of different familys, be that the family selective breeding and plasm resource protection of Pacific oyster has important
Using value.
A kind of method suitable for Pacific oyster Parentage determination, comprises the following steps that：
1）In the snp database that Illumina GoldenGate methods are obtained, a class and two class SNP mutation positions are searched for
Point, described mutational site is C/T＆G/A and C/A＆G/T；
2）The genotype data library file of filial generation and parent is set up, using Cervus3.0 softwares the something lost of each SNP site is obtained
Pass parameter；
3）Using Cervus3.0 softwares, family simulation identification and analysis are carried out according to the gene frequency in each site, then
Carry out actual Parentage determination analysis, calculate the LOD value of candidate parent couple, select LOD value highest and be on the occasion of parent to conduct
Real Parent, calculates Parentage determination success rate.
Further, the step of described actual Parentage determination is analyzed includes the DNA for extracting parent and filial generation, builds PCR anti-
System is answered, in Roche LightCycler480 HRM analyses are carried out.
Further, the PCR primer in described actual Parentage determination analysis is any pair of in table 1.
Further, the PCR reaction systems in described actual Parentage determination analysis are 10 μ l：The μ L of 5ng/ μ L template DNAs 1,
Roche HRM Master5 μ L, 25ng/ul MgCl21.2-1.4 μ L, each 0.2 μ L surpluses distilled water of 10ng/ul primers is mended
Foot；PCR amplification conditions are：94 DEG C of denaturations 10min, 94 DEG C of degeneration 10s, 62-55 DEG C of annealing 10s, 72 DEG C of extension 5s, totally 35
Circulation, last 72 DEG C of extensions 10min.
Further, the screening technique of PCR primer in described actual Parentage determination analysis：
1）In the snp database that Illumina GoldenGate methods are obtained, a class and two class SNP mutation positions are searched for
Point（C/T＆G/A, C/A＆G/T）, the flanking sequence in site utilizes Primer3.0（http://primer3.ut.ee/）Design
Primer；Avoid the appearance of hairpin structure, primer dimer during design primer as far as possible；2）Individuality is extracted using Qiagen test kits too
Flat ocean Concha Ostreae genomic DNA, with PicoGreen fluorescent dyes test kit the quantitative analyses of DNA are carried out, and sample DNA concentration is adjusted
It is whole to 5ng/ μ L, for the screening and optimization of SNP marker；
3）Do template with the DNA after quantitative carries out HRM detections using Roche LightCycler480, by changing annealing
Temperature and Mg2+Concentration screening goes out amplification efficiency height, solubility curve clearly primer, and obtains the PCR reaction systems of optimum and draw
The optimum annealing temperature of thing.
Present invention beneficial effect compared with prior art：
The present invention develops a kind of method suitable for Pacific oyster Parentage determination, and optimize its reaction system and
PCR amplification conditions, the experiment proved that the features such as these primers have high reproducible, stability, solubility curve high resolution, can
The family paternity identification analysis of Pacific oyster is applied to, is that the family selective breeding of Pacific oyster lays the foundation.
Description of the drawings
Fig. 1：191-153 sites allelic is in parent and the heredity of filial generation；
Fig. 2：The LOD value of parent's identification.
Specific embodiment
Technical scheme is further explained below by embodiment, but protection scope of the present invention is not by real
Apply any pro forma restriction of example.
Embodiment 1
1）Design of primers and optimization.
In the snp database that Illumina GoldenGate methods are obtained, a class and two class SNP mutation sites are searched for
（C/T＆G/A, C/A＆G/T）, the flanking sequence in site utilizes Primer3.0（http://primer3.ut.ee/）Design is drawn
Thing.Avoid the appearance of hairpin structure, primer dimer during design primer as far as possible.Primer length is 18-30bp, and annealing temperature is
55-65 DEG C, expanding fragment length is 50-180bp.6 individual Pacific oyster genomes are extracted using Qiagen test kits
DNA, with PicoGreen fluorescent dyes test kit the quantitative analyses of DNA are carried out, and sample DNA concentration is adjusted to 5ng/ μ L, are used for
The screening and optimization of SNP marker.Do template with the DNA after quantitative carries out HRM detections using Roche LightCycler480, leads to
Cross change annealing temperature and Mg2+Concentration screening goes out amplification efficiency height, solubility curve clearly primer, and it is anti-to obtain the PCR of optimum
Answer the optimum annealing temperature of system and primer.10 μ l PCR reaction systems are as follows：5ng/ μ L template DNAs 1 μ L, Roche HRM
Master5 μ L, 25ng/ulMgCl21.4 μ L, each 0.2 μ L of 10ng/ul primers, surplus distilled water is supplied.PCR amplification conditions
For：94 DEG C of denaturations 10min, 94 DEG C of degeneration 10s, 62-55 DEG C of annealing 10s, 72 DEG C of extension 5s, totally 35 circulations, last 72 DEG C
Extend 10min.
2）Concha Ostreae extracting genome DNA and quantitative.
Genomic DNA is extracted to all 18 candidate parents and the alternative Qiagen test kits of 78 sons, using PicoGreen
Fluorescent dye test kit carries out the quantitative analyses of DNA, and to 5ng/ μ L, 4 DEG C preserve for Parentage determination analysis adjustment final concentration.
3）Structure based on the SNP Parentage determination systems of HRM
HRM analyses are carried out in Roche LightCycler480,10 μ l PCR reaction systems are as follows：5ng/ μ L template DNAs 1
μ L, Roche HRM Master5 μ L, 25ng/ul MgCl21.4 μ L, each 0.2 μ L of 10ng/ul primers, surplus distilled water is mended
Foot.PCR reaction conditions are：94 DEG C of denaturations 10min, 94 DEG C of degeneration 10s, Ta（The optimum annealing temperature of each primer optimization）Annealing
10s, 72 DEG C of extension 5s, totally 35 circulations, last 72 DEG C of extensions 10min.According to mendelian inheritance and feature solubility curve
Shape, filter out the SNP primers that 38 pairs are reproducible, stability is high, homozygote and heterozygote gene can be clearly distinguished
Type（Such as Fig. 1）, primer details are shown in Table 1, are applied to Parentage determination and the analysis of Pacific oyster.
4) the paternity identification analysis of Pacific oyster family
Set up filial generation（n=78）And parent（N=18）Genotype data library file, in Illumina GoldenGate sides
In the snp database that method is obtained, a class and two class SNP mutation sites are searched for, using Cervus3.0 softwares each SNP site is obtained
Genetic parameter, including the information such as number of alleles, heterozygosity, polymorphism information content, probability of exclusion and accumulation probability of exclusion（See
Table 2）.Using Cervus3.0 softwares, family simulation identification and analysis are carried out according to the gene frequency in each site, then carried out
Actual Parentage determination analysis.Described actual Parentage determination analysis is comprised the following steps that：Using step 2）DNA after quantitative,
Roche LightCycler480 carry out HRM analyses, PCR reaction systems：10 μ l PCR reaction systems are as follows：5ng/ μ L templates
DNA1 μ L, Roche HRM Master5 μ L, 25ng/ul MgCl21.4 μ L, each 0.2 μ L of 10ng/ul primers（Primer is _ F-
It is any pair of in TCTCATCCTCATCCAAGTC, R-CACACGTGTAAGAAAGGATG, or the primer of table 1）, surplus
Distilled water is supplied.PCR reaction conditions are：94 DEG C of denaturations 10min, 94 DEG C of degeneration 10s, 56 DEG C of annealing 10s, 72 DEG C of extension 5s,
Totally 35 circulations, last 72 DEG C of extensions 10min.
Calculate the LOD value of optimal candidate parent couple（See Fig. 2）, select LOD value highest and be on the occasion of parent to as true
Positive Parent, calculates Parentage determination success rate.As a result show, 93.2% individuality has successfully been assigned in 9 familys, in addition,
Have 6.8% it is individual not specified to correct parent, probably due to caused by parental animal disappearance.
The primer information of 38 SNP markers of the Pacific oyster Parentage determination system of table 1
Genetic parameters estimate result of 238 SNP sites of table in Pacific oyster family
Claims (3)
1. it is a kind of to be applied to Pacific oyster Parentage determination method, it is characterised in that to comprise the following steps that：
1) in the snp database that Illumina GoldenGate methods are obtained, a class and two class SNP mutation sites, institute are searched for
The mutational site stated is C/T＆G/A and C/A＆G/T；
2) the genotype data library file of filial generation and parent is set up, using Cervus3.0 softwares the heredity ginseng of each SNP site is obtained
Number；
3) using Cervus3.0 softwares, family simulation identification and analysis are carried out according to the gene frequency in each site, is then carried out
The analysis of actual Parentage determination, calculates the LOD value of candidate parent couple, select LOD value highest and be on the occasion of parent to as real
Parent, calculates Parentage determination success rate；
The step of described actual Parentage determination is analyzed includes the DNA for extracting parent and filial generation, builds PCR reaction systems,
Roche LightCycler480 carry out HRM analyses；
PCR primer in described actual Parentage determination analysis is any pair of in table 1；
The primer information of 38 SNP markers of the Pacific oyster Parentage determination system of table 1
2. it is according to claim 1 a kind of suitable for Pacific oyster Parentage determination method, it is characterised in that described reality
PCR reaction systems in the Parentage determination analysis of border are 10 μ L：The μ L of 5ng/ μ L template DNAs 1, Roche HRM Master5 μ L,
25ng/ul MgCl21.2～1.4 μ L, each 0.2 μ L surplus distilled waters of 10ng/ul primers are supplied；PCR amplification conditions are：94℃
Denaturation 10min, 94 DEG C of degeneration 10s, 62～55 DEG C of annealing 10s, 72 DEG C of extension 5s, totally 35 circulations, last 72 DEG C of extensions
10min。
3. it is according to claim 1 a kind of suitable for Pacific oyster Parentage determination method, it is characterised in that described reality
The screening technique of PCR primer in the Parentage determination analysis of border：
1) in the snp database that Illumina GoldenGate methods are obtained, a class and two class SNP mutation sites, institute are searched for
The mutational site stated is C/T＆G/A and C/A＆G/T, and the flanking sequence in site designs primer using Primer3.0；Design primer
Shi Jinliang avoids the appearance of hairpin structure, primer dimer；
2) individual Pacific oyster genomic DNA is extracted using Qiagen test kits, is entered with PicoGreen fluorescent dye test kits
The quantitative analyses of row DNA, sample DNA concentration are adjusted to 5ng/ μ L, for the screening and optimization of SNP marker；
3) do template with the DNA after quantitative carries out HRM detections using Roche LightCycler480, by changing annealing temperature
And Mg2+Concentration screening goes out that amplification efficiency is high, solubility curve clearly primer, and obtains the PCR reaction systems and primer of optimum
Optimum annealing temperature.
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104152444B (en) * | 2014-07-24 | 2017-09-29 | 中国科学院海洋研究所 | A kind of SNP marker related to long oyster glycogen content character and its application |
| CN105002293A (en) * | 2015-08-18 | 2015-10-28 | 中国水产科学研究院黑龙江水产研究所 | Method utilizing microsatellite markers to identify polycultured Songpu mirror carp family |
| CN107475413B (en) * | 2017-09-20 | 2021-05-28 | 中国科学院海洋研究所 | Method for screening crassostrea gigas parent shellfish with high content of unsaturated fatty acid C20:3 omega 6 |
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