CN104694660A - Oval pompanos trevally family paternity test method - Google Patents

Abstract

The invention discloses an oval pompanos trevally family paternity test method. For an embodiment for preparing mixed families through multiple parents, 11 pairs of micro-satellite primers which are high in genetic diversity and stable in amplification efficiency are screened out. The purpose of detecting the individual parent-child relationship of an oval pompanos trevally family is achieved in combination with capillary electrophoresis sequencing and software analysis. By means of the method, the oval pompanos trevally paternity test accuracy rate can reach over 90%.

Description

The method of Trachinotus ovatus family paternity test

Technical field

The present invention relates to fish breeding and molecular marking technique field, be specifically related to a kind of method utilizing microsatellite molecular marker qualification Trachinotus ovatus to raise together with family progeny parent.

Background technology

Trachinotus ovatus is subordinate to Perciformes (Perciformes), Scad section (Carangidae), silvery pomfret Scad subfamily (Trachinotinae), silvery pomfret Scad belongs to (Trachinotus), be commonly called as golden silvery pomfret, yellow cured silvery pomfrets etc., are one warm water fisheses at the middle and upper levels, are distributed widely in the torrid zone, subtropical seas, there is fast growth, the features such as meat is tender delicious, and nutritive value is high, have now become the Important Economic kind of coastal areas of southern China pond and cage culture.

In the last few years, Trachinotus ovatus market demand significantly rises, become the most large kind of southern marine fish, current China gold silvery pomfret marketable fish annual production more than 200,000 tons, only just there are 500 tons of chilled Trachinotus ovatus trading volumes Zhanjiang fish trade market every day.Market large so is also very huge to the demand of Trachinotus ovatus seed.Trachinotus ovatus is water warm seawater fish, and Trachinotus ovatus mainly concentrates on the ground such as Hainan, Guangdong, Fujian by temperature conditions culturing area.And Hainan is located in subtropics, torrid areas, seawater temperature is relatively high, the hatching of suitable Trachinotus ovatus and nursery.At present, three provinces and regions all Trachinotus ovatus cultivation seeds all come from Hainan Region.Due to the inbred phenomenon of Trachinotus ovatus ubiquity, cause seed kind matter to be degenerated, poor growth, the problems such as disease outburst highlight day by day.The domestic pattern of man of wild origin that tradition seed breeding industry adopts is faced with huge challenge, carries out fine-variety breeding just become the extremely urgent task of aquaculture investigator for Trachinotus ovatus.

Family selective breeding is the important foundation means of fish breeding, be obviously be better than its relatives according to certain or a few proterties, production performance is significantly higher than its relatives is mixed with and selects some defect individuals in dissimilar primitive horde and reserve seed for planting, set up several or several familys and raise up seed, by generation compared with original population and check variety, selecting and remain, those meet the excellent system of original selective goal, and then carry out strain performance measurement.In fish breeding, family selective breeding is particularly important, and nearly all incubation kind all needs through this step of family selective breeding.The foundation of current Technique in Fishes family adopts 1 (female) to 1 (hero) mating production filial generation usually.The family comparatively small amt that such mode is set up, and each family of management is affected by environment comparatively large separately, unfavorable to families selecting; And individual physical markings is carried out to mixed family, very difficult when more family.Judgement at present for Trachinotus ovatus breeding ripening degree and male and female sex is more difficult, also and inapplicable 1 to 1 the mode of production.Therefore be insoluble problem for the foundation of Trachinotus ovatus family and the qualification of family always, the Trachinotus ovatus breeding work hindered to a great extent.

Microsatellite DNA (Microsatellites DNA) is a kind of moderately repetitive sequence be distributed widely in eukaryotic gene group.Microsatellite marker is with its rich polymorphism, and the advantage of codominant inheritance is widely used in population genetic variations analysis, character analysis, Idioplasm identification, Parentage determination, the fields such as genetic mapping.Micro-satellite genetic information of parent can pass to filial generation by breeding, micro-satellite labeling technique by detect different parent-offspring's individuality micro-satellite difference discriminate individuals belonging to family.Have not yet to see report microsatellite marker being used for Trachinotus ovatus Parentage determination.

Summary of the invention

The object of this invention is to provide a kind of method utilizing microsatellite marker to identify Trachinotus ovatus family, for Trachinotus ovatus breeding provides the molecule marker instrument of paternity test.

To achieve these goals, technical scheme of the present invention is: the method providing the paternity test of a kind of Trachinotus ovatus family, the method comprises the steps:

1) select Trachinotus ovatus build comparatively large, parent, as parent, is mixed into row artificial propagation by the speed of growth faster individual 87 tails; When fry grows to 10 centimeters, random selecting 10,000 tail proceeds to cage culture, as the family colony for paternity test;

2) selecting step 1) Trachinotus ovatus breeding parent isozyme, extract genomic dna, take DNA as template, select 51 pairs of micro-satellite primers to carry out nested PCR amplification;

3) step 2) described in nested PCR amplification method refer in PCR system, comprise 3 primers: locus specificity forward primer, its 5 ' end adds M13 sequence (5 '-CAGTCGGGCGTCATCA-3 '); Locus specificity reverse primer; The fluorescently-labeled M13 universal primer of FAM, PET, VIC, NED tetra-kinds; PCR reaction system 20ul:50ng DNA, 2ul dNTP (2.5mM), 2ul10 × Buffer mixture, 1U rTaq, 0.4ul containing the forward primer (10mM) adding universal primer M13,0.5ul reverse primer, the fluorescent mark universal primer of 0.1ul, ddH ₂o supplies;

4) Trachinotus ovatus parent in the PCR primer of 51 microsatellite locus through ABI3730 type Genetic Analyser, gene type assay is carried out according to fluorescent mark, use the analysis of Genemapper3.7 software simulation, filter out and be applicable to effective microsatellite locus 11 that qualification Trachinotus ovatus raises together with family: ZD13, ZD15, TBG008, ZD02, ZD03, ZD04, ZD11, ZD12, ZD01, ZD10, TBG016;

5) Trachinotus ovatus 10,000 filial generations were raised together with for 7 monthly ages, random selecting 2000 tail is individual, electronic marker is implanted and each individual isozyme of clip at the muscle of back of every bar fish, extract genes of individuals group DNA, selecting step 4) in 11 effective microsatellite locus, analyze the genotype of filial generation;

6) according to parent and the filial generation genotype at 11 microsatellite locus, PAPA 2.0 software is adopted to distinguish different filial generation, the Parent of qualification offspring individual.

Wherein step 4) described in the nucleotide sequence of the effective micro-satellite primers of 11 couple as follows.

Table 1 Trachinotus ovatus paternity test micro-satellite primers

* respectively synthesize four kinds of fluorescently-labeled M13 primers, with Trachinotus ovatus primer with the use of

The present invention has the following advantages:

1. the mode that the present invention adopts parent fish population to breed prepares mixed family, without the need to manually matching breeding one to one.Can be used for male and female sex not to be easily distinguishable and the uppity Trachinotus ovatus artificial propagation of ripening degree and family breeding build, solve the problem of this type fish family fine-variety breeding greatly.

2. in the present invention, the filial generation of mixed family cultivates from zygote under this one-phase of marketable fish is all in same environment, can avoid the error that independent family breeding environment difference causes, and improves the accuracy of family selective breeding.

3. the present invention adopts mixing to cultivate the mode of family, does not need to maintain huge isolation breeding facility, greatly save management and aquaculture cost from nursery to half adult fish stage.

4. the present invention adopts nested PCR amplification method, FAM, PET, VIC, NED tetra-kinds of fluorescent marks are selected to mark universal primer M13, adopt micro-satellite forward primer 5 ' to hold and add the method for M13 sequence, for qualification individual more time, greatly can save the fluorescently-labeled expense of primer.

Accompanying drawing explanation

The pcr amplification result of Fig. 1 primer TBG008;

The pcr amplification result of Fig. 2 primer ZD15;

Fig. 3 ZD02 site allelic go out peak figure;

Fig. 4 ZD03 site allelic go out peak figure;

Embodiment

1) Trachinotus ovatus family breed and filial generation cultivation

From Trachinotus ovatus natural population (Hainan Region), select 87 tail defect individuals as parent, implant electronic marker at its muscle of back and measure the growth traits data of each individuality.Every bar parent clip one fritter fin ray, is stored in 95% alcohol, is stored in-20 DEG C.Parent carries out artificial induced spawning by the method for injecting hormone, and 87 tail parent populations are all injected, the method for injection hormone: disposable injection chorionic-gonadotropin hormone (HCG), injected dose: 200IU/kg; Luteotropin releasing hormone d-ala analog (LHRH-A2), injected dose: 3ug/kg.87 tail parent population common property 10kg zygotes, getting 1.2kg zygote, to put into area be that the pond of 4 mu is hatched, and estimate after prelarva 1,120,000 tail cultivates 60 days after 1d, prelarva mean length is 10 centimeters, and random selecting 10,000 proceeds to 6m × 6m cage culture.Proceed to another 6m × 6m cage culture from 10,000 middle random selecting 2200 tails and squeeze into electronic marker when cultivating for 7 monthly age, Taking Pictures recording growth traits clip each filial generation tail fin tissue, being stored in 95% alcohol, being stored in-20 DEG C.Ammonium acetate method is adopted to extract the genomic dna of parent and family individuality.

2) screening of PCR primer and amplification reaction system

Have chosen 51 Trachinotus ovatus microsatellite locus and carry out pcr amplification, most site expanding effect not good or in Trachinotus ovatus colony difference less, it is high that finishing screen selects 11 pairs of genetic polymorphisms, the stable primer of amplification efficiency is used for Trachinotus ovatus paternity test, 11 to effective microsatellite marker amplimer and condition as shown in table 1.PCR reaction system is 20ul:10 × PCR Buffer 2ul, dNTP mixture (2.5mM each) 2ul, TaKaRa Taq 0.2ul (5U/ul) (precious biotechnology Dalian company limited produces), 10umol/L forward primer 5 ' end adds M13 universal primer 0.4ul, 10umol/L reverse primer 0.5ul, 10umol/L M13 fluorescent primer 0.1ul.Amplified reaction completes in ABI2720PCR system, and PCR program is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s; Tm anneals 30s; 72 DEG C extend 30s, and reaction carries out 32 circulations, 94 DEG C of sex change 30s; 53 DEG C of annealing 30s; 72 DEG C extend 30s, and 8 circulations are carried out in reaction; Finally extend 10min at 72 DEG C.Wherein Tm value sets according to table 1 different primers annealing temperature.The amplification of Fig. 1, Fig. 2 exposition microsatellite locus.

3) microsatellite locus gene type assay

After PCR has reacted, post to physico-chemical analysis center, Beijing and carry out upper machine testing, detecting instrument used is ABI3730 Genetic Analyser.Do gene type assay with software Gene Mapper3.7, read parent and filial generation microsatellite locus genotype respectively.Fig. 3, Fig. 4 are the peak figure of moiety site.

4) effectively microsatellite locus choose and Trachinotus ovatus paternity test is analyzed

Adopt step 4) methods analyst 87 tail Trachinotus ovatus parent and 2200 odd amount in addition to the round numbers generation in the genotype of 51 pairs of microsatellite locus, by genotype and the parent genotype matching relationship of unknown parent's sex module simulation multiplication family filial generation in software PAPA 2.0, screen 11 microsatellite locus (ZD13, ZD15, TBG008, ZD02, ZD03, ZD04, ZD11, ZD12, ZD01, ZD10, TBG016) paternity test accuracys rate and can arrive 90.625% (table 2).

5) interpretation of result

Raise together with 96 tails in family progeny at 11 microsatellite locus to 87 tail Trachinotus ovatus parents and 2200 tails to increase and gene type.Genotype application Gene Mapper 3.7 software is analyzed, and Trachinotus ovatus paternity test application PAPA 2.0 software unknown parent's sex module is analyzed.For ensureing the accuracy of experimental result, only all the match is successful at guaranteed all micro-satellite seats, and meet the just confirmation parent child relationship of parent's mating system.Final confirmation 87 tail raises together with the father and mother of filial generation, and paternity test rate is 90.625%.

The individual paternity test information table of table 2 Trachinotus ovatus 96 tail

Above disclosedly be only preferred embodiment of the present invention, certainly can not limit the interest field of the present invention with this, therefore according to the equivalent variations that the claims in the present invention are done, still belong to the scope that the present invention is contained.

Claims (1)

1. a method for Trachinotus ovatus family paternity test, the method comprises the steps:

1) select Trachinotus ovatus build comparatively large, parent, as parent, is mixed into row artificial propagation by the speed of growth faster individual 87 tails; When fry grows to 10 centimeters, random selecting 10,000 tail proceeds to cage culture, as the family colony for paternity test;

2) selecting step 1) Trachinotus ovatus breeding parent isozyme, extract genomic dna, take DNA as template, select 51 pairs of micro-satellite primers to carry out nested PCR amplification;

3) step 2) described in nested PCR amplification method refer in PCR system, comprise 3 primers: locus specificity forward primer, its 5 ' end adds M13 sequence (5 '-CAGTCGGGCGTCATCA-3 '); Locus specificity reverse primer; The fluorescently-labeled M13 universal primer of FAM, PET, VIC, NED tetra-kinds; PCR reaction system 20ul:50ng DNA, 2ul dNTP (2.5mM), 2ul10 × Buffer mixture, 1U rTaq, 0.4ul containing the forward primer (10mM) adding universal primer M13,0.5ul reverse primer, the fluorescent mark universal primer of 0.1ul, ddH ₂o supplies;

4) Trachinotus ovatus parent in the PCR primer of 51 microsatellite locus through ABI3730 type Genetic Analyser, gene type assay is carried out according to fluorescent mark, use the analysis of Genemapper3.7 software simulation, filter out and be applicable to effective microsatellite locus 11 that qualification Trachinotus ovatus raises together with family: ZD13, ZD15, TBG008, ZD02, ZD03, ZD04, ZD11, ZD12, ZD01, ZD10, TBG016;

5) Trachinotus ovatus 10,000 filial generations were raised together with for 7 monthly ages, random selecting 2000 tail is individual, electronic marker is implanted and each individual isozyme of clip at the muscle of back of every bar fish, extract genes of individuals group DNA, selecting step 4) in 11 effective microsatellite locus, analyze the genotype of filial generation;

6) according to parent and the filial generation genotype at 11 microsatellite locus, PAPA 2.0 software is adopted to distinguish different filial generation, the Parent of qualification offspring individual.