CN103605913A - Method applied to identification of pacific oyster family - Google Patents

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

A kind of method that is applicable to Pacific oyster Parentage determination

Technical field

The invention belongs to genome molecule labelling technique field, relate to a kind of method that is applicable to Pacific oyster Parentage determination.

Background technology

Oyster is subordinate to Mollusca, Bivalvia, pearl shell order, and its meat flavour is delicious, nutritious, just by the mankind, is eaten from the ancient times, has very high economic worth and medical value.Oyster is worldwide distribution monoid, has found that at present nearly all there is production in the 100Duo Zhong， world country that respectively borders on the sea, is the economic shellfish of current China and world wide production maximum.

As the large cultivated shellfish of the first in the world, the genetic improvement of oyster is always subject to domestic and international expert and scholar's generally attention, and has carried out a large amount of research work.Although China is oyster culture big country, be not also oyster culture power, there is the problems such as genetic improvement research relatively lags behind, Study on Genetic Basis relative thin is weak, breeding is deficient, had a strong impact on and restricted healthy aquaculture and the genetic improvement work of China oyster.It is the important means that keeps good economic characters and obtain improved seeds and strain that family is selected breeding, widespread use in animals and plants.Complete, pedigree information can be avoided inbreeding, keep the genetic diversity of species, to promoting the genetic improvement work of shellfish to have important realistic meaning accurately.SNP(single nucleotide polymorphism) refer to the single nucleotide variation on DNA sequence dna, it is the third generation molecular genetic marker after microsatellite marker, there is genome and distribute extensively, be easy to the advantages such as detection, high genetic stability, in Parentage determination, have obvious advantage.High-resolution fusion curve (HRM) is a kind of SNP somatotype new technology of analyzing of dissolving based on high resolving power, has high flux, low cost, easy-operating feature, has been widely used in the research in the fields such as science of heredity, genomics, biology.Utilize modern molecular labeling new technology; set up a set of SNP Parentage determination system that is applicable to Pacific oyster; will be for determining that between individuality, sibship provides effective way; for avoiding inbreeding depression that strong scientific basis is provided, for the family selective breeding of oyster, build the research work such as inbred strais, plasm resource protection and lay the foundation.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of method that is applicable to Pacific oyster Parentage determination, contributes to fast, accurately to differentiate the Pacific oyster of different familys, for family selective breeding and the plasm resource protection of Pacific oyster has important using value.

A method that is applicable to Pacific oyster Parentage determination, concrete steps are as follows:

1) in the snp database obtaining in Illumina GoldenGate method, search for a class and two class SNP mutational sites, described mutational site is C/T & G/A and C/A & G/T;

2) set up filial generation and parent's genotype data library file, utilize Cervus3.0 software to obtain the genetic parameter in each SNP site;

3) utilize Cervus3.0 software, according to the gene frequency in each site, carry out family simulation identification and analysis, then carry out actual Parentage determination analysis, the LOD value that calculated candidate parent is right, select LOD value the highest and be on the occasion of parent to as real Parent, calculating Parentage determination success ratio.

Further, the step of described actual Parentage determination analysis comprises the DNA that extracts parent and filial generation, builds PCR reaction system, at Roche LightCycler480, carries out HRM analysis.

Further, the PCR primer in described actual Parentage determination analysis is any a pair of in table 1.

Further, the PCR reaction system in described actual Parentage determination analysis is 10 μ l:5ng/ μ L template DNA 1 μ L, Roche HRM Master5 μ L, 25ng/ul MgCl ₂1.2-1.4 μ L, each 0.2 μ L surplus distilled water of 10ng/ul primer is supplied; Pcr amplification condition is: 94 ℃ of denaturation 10min, and 94 ℃ of sex change 10s, 62-55 ℃ of annealing 10s, 72 ℃ are extended 5s, totally 35 circulations, last 72 ℃ are extended 10min.

Further, the screening technique of PCR primer in described actual Parentage determination analysis:

1) in the snp database obtaining in Illumina GoldenGate method, search for a class and two class SNP mutational site (C/T & G/A, C/A & G/T), at the flanking sequence in site, utilize Primer3.0(http: //primer3.ut.ee/) design primer; During design primer, avoid the appearance of hairpin structure, primer dimer as far as possible; 2) utilize Qiagen kit to extract individual Pacific oyster genomic DNA, with PicoGreen fluorescent dye kit, carry out the quantitative test of DNA, sample DNA concentration is adjusted to 5ng/ μ L, for screening and the optimization of SNP mark;

3) with the DNA after quantitative, do template and use Roche LightCycler480 to carry out HRM detection, by changing annealing temperature and Mg ²⁺concentration screening goes out that amplification efficiency is high, solubility curve primer clearly, and obtains optimum PCR reaction system and the optimum annealing temperature of primer.

The present invention's beneficial effect compared with prior art:

The present invention develops a kind of method that is applicable to Pacific oyster Parentage determination, and its reaction system and pcr amplification condition have been optimized, the experiment proved that these primers have reproducible, stability is high, solubility curve resolution high, the family paternity identification that can be applicable to Pacific oyster is analyzed, for the family selective breeding of Pacific oyster lays the foundation.

Accompanying drawing explanation

Fig. 1: in 191-153 site, allele is in the heredity of parent and filial generation;

Fig. 2: the LOD value that parent identifies.

Embodiment

Below by embodiment, technical scheme of the present invention is further explained, but protection scope of the present invention is not subject to any pro forma restriction of embodiment.

Embodiment 1

1) design of primers and optimization.

In the snp database obtaining in Illumina GoldenGate method, search for a class and two class SNP mutational site (C/T & G/A, C/A & G/T), at the flanking sequence in site, utilize Primer3.0(http: //primer3.ut.ee/) design primer.During design primer, avoid the appearance of hairpin structure, primer dimer as far as possible.Primer length is 18-30bp, and annealing temperature is 55-65 ℃, and expanding fragment length is 50-180bp.Utilize Qiagen kit to extract 6 individual Pacific oyster genomic DNAs, with PicoGreen fluorescent dye kit, carry out the quantitative test of DNA, sample DNA concentration is adjusted to 5ng/ μ L, for screening and the optimization of SNP mark.With the DNA after quantitative, do template and use Roche LightCycler480 to carry out HRM detection, by changing annealing temperature and Mg ²⁺concentration screening goes out that amplification efficiency is high, solubility curve primer clearly, and obtains optimum PCR reaction system and the optimum annealing temperature of primer.10 μ l PCR reaction systems are as follows: 5ng/ μ L template DNA 1 μ L, Roche HRM Master5 μ L, 25ng/ulMgCl ₂1.4 μ L, each 0.2 μ L of 10ng/ul primer, surplus distilled water is supplied.Pcr amplification condition is: 94 ℃ of denaturation 10min, and 94 ℃ of sex change 10s, 62-55 ℃ of annealing 10s, 72 ℃ are extended 5s, totally 35 circulations, last 72 ℃ are extended 10min.

2) oyster extracting genome DNA and quantitative.

All 18 candidate parents and 78 sons are substituted to Qiagen kit extraction genomic DNA, utilize PicoGreen fluorescent dye kit to carry out the quantitative test of DNA, adjust final concentration to 5ng/ μ L, 4 ℃ of preservations are for Parentage determination analysis.

3) structure of the SNP Parentage determination system based on HRM

At Roche LightCycler480, carry out HRM analysis, 10 μ l PCR reaction systems are as follows: 5ng/ μ L template DNA 1 μ L, Roche HRM Master5 μ L, 25ng/ul MgCl ₂1.4 μ L, each 0.2 μ L of 10ng/ul primer, surplus distilled water is supplied.PCR reaction conditions is: 94 ℃ of denaturation 10min, 94 ℃ of sex change 10s, the optimum annealing temperature that each primer of Ta(is optimized) annealing 10s, 72 ℃ are extended 5s, totally 35 circulations, last 72 ℃ are extended 10min.According to the shape of mendelian inheritance and feature solubility curve, filter out 38 pairs of SNP primers reproducible, stability is high, can clearly distinguish homozygote and heterozygote genotype (as Fig. 1), primer details, 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's (N=18) genotype data library file, in the snp database obtaining in Illumina GoldenGate method, search for a class and two class SNP mutational sites, utilize Cervus3.0 software to obtain the genetic parameter in each SNP site, comprise the information (in Table 2) such as number of alleles, heterozygosity, polymorphism information content, probability of exclusion and accumulation probability of exclusion.Utilize Cervus3.0 software, according to the gene frequency in each site, carry out family simulation identification and analysis, then carry out actual Parentage determination analysis.It is as follows that described actual Parentage determination is analyzed concrete steps: utilize step 2) DNA after quantitatively, at Roche LightCycler480, carry out HRM analysis, PCR reaction system: 10 μ l PCR reaction systems are as follows: 5ng/ μ L template DNA 1 μ L, Roche HRM Master5 μ L, 25ng/ul MgCl ₂1.4 μ L, each 0.2 μ L(primer of 10ng/ul primer is _ F-TCTCATCCTCATCCAAGTC, R-CACACGTGTAAGAAAGGATG can be also any a pair of in table 1 primer), surplus distilled water is supplied.PCR reaction conditions is: 94 ℃ of denaturation 10min, and 94 ℃ of sex change 10s, 56 ℃ of annealing 10s, 72 ℃ are extended 5s, totally 35 circulations, last 72 ℃ are extended 10min.

Calculate the right LOD value (see figure 2) of optimal candidate parent, select LOD value the highest and be on the occasion of parent to the real Parent of conduct, calculating Parentage determination success ratio.Result shows, 93.2% individuality has successfully been assigned in 9 familys, in addition, have 6.8% individuality not to be assigned to correct parent, may be because parent's sample disappearance cause.

The primer information of 38 SNP marks of table 1 Pacific oyster Parentage determination system

The genetic parameters estimate result of a table 238 SNP site in Pacific oyster family

Claims (5)

1. be applicable to a Pacific oyster Parentage determination method, it is characterized in that concrete steps are as follows:

1) in the snp database obtaining in Illumina GoldenGate method, search for a class and two class SNP mutational sites, described mutational site is C/T & G/A and C/A & G/T;

2) set up filial generation and parent's genotype data library file, utilize Cervus3.0 software to obtain the genetic parameter in each SNP site;

3) utilize Cervus3.0 software, according to the gene frequency in each site, carry out family simulation identification and analysis, then carry out actual Parentage determination analysis, the LOD value that calculated candidate parent is right, select LOD value the highest and be on the occasion of parent to as real Parent, calculating Parentage determination success ratio.

2. a kind of Pacific oyster Parentage determination method that is applicable to according to claim 1, the step that it is characterized in that described actual Parentage determination analysis comprises the DNA that extracts parent and filial generation, build PCR reaction system, at Roche LightCycler480, carry out HRM analysis.

3. a kind of Pacific oyster Parentage determination method that is applicable to according to claim 2, is characterized in that PCR primer in described actual Parentage determination analysis is any a pair of in table 1.

4. a kind of Pacific oyster Parentage determination method that is applicable to according to claim 2, it is characterized in that the PCR reaction system in described actual Parentage determination analysis is 10 μ L:5ng/ μ L template DNA 1 μ L, Roche HRM Master5 μ L, 25ng/ul MgCl ₂1.2～1.4 μ L, each 0.2 μ L surplus distilled water of 10ng/ul primer is supplied; Pcr amplification condition is: 94 ℃ of denaturation 10min, and 94 ℃ of sex change 10s, 62～55 ℃ of annealing 10s, 72 ℃ are extended 5s, totally 35 circulations, last 72 ℃ are extended 10min.

5. a kind of Pacific oyster Parentage determination method that is applicable to according to claim 2, it is characterized in that described actual Parentage determination analyze in the screening technique of PCR primer:

1) in the snp database obtaining in Illumina GoldenGate method, search for a class and two class SNP mutational sites, described mutational site is C/T & G/A and C/A & G/T, and the flanking sequence in site utilizes Primer3.0 design primer; During design primer, avoid the appearance of hairpin structure, primer dimer as far as possible;

2) utilize Qiagen kit to extract individual Pacific oyster genomic DNA, with PicoGreen fluorescent dye kit, carry out the quantitative test of DNA, sample DNA concentration is adjusted to 5ng/ μ L, for screening and the optimization of SNP mark;

3) with the DNA after quantitative, do template and use Roche LightCycler480 to carry out HRM detection, by changing annealing temperature and Mg ²⁺concentration screening goes out that amplification efficiency is high, solubility curve primer clearly, and obtains optimum PCR reaction system and the optimum annealing temperature of primer.

Images