CN104694660B - The method of egg-shaped pompano family paternity test - Google Patents
The method of egg-shaped pompano family paternity test Download PDFInfo
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Abstract
The invention discloses a kind of method of egg-shaped pompano family paternity test, for the example that mixed family is prepared using multi-parent strain, 11 pairs of micro-satellite primers that genetic diversity is high, amplification efficiency is stable are filtered out.With reference to Capillary Electrophoresis order-checking and software analysis, the purpose of detection egg-shaped pompano family individuality parent child relationship is reached.By the method for the present invention, egg-shaped pompano paternity test accuracy rate can reach more than 90%.
Description
Technical field
The present invention relates to fish breeding and molecular marking technique field, and in particular to one kind is using microsatellite molecular marker mirror
Determine the method that egg-shaped pompano raises together with family progeny parent.
Background technology
Egg-shaped pompano is subordinate to Perciformes (Perciformes), Scad sections (Carangidae), silvery pomfret Scad subfamilies
(Trachinotinae), silvery pomfret Scad category (Trachinotus), is commonly called as golden silvery pomfret, yellow cured silvery pomfret etc., is a kind of warm water fishes at the middle and upper levels,
It is distributed widely in the torrid zone, subtropical seas, with fast growth, meat tenderness is delicious, the features such as being of high nutritive value, as me
The coastal pond in state south and the Important Economic kind of cage culture.
In the last few years, egg-shaped pompano market demand significantly rose, and had become the most large of southern marine fish
Species, more than 200,000 tons of current China's gold silvery pomfret marketable fish annual production, just there are 500 tons of ice only Zhanjiang fish trade market daily
Fresh egg-shaped pompano trading volume.So big market is also very huge to the demand of egg-shaped pompano seed.Egg-shaped pompano is
Water warm seawater fish, egg-shaped pompano is concentrated mainly on the ground such as Hainan, Guangdong, Fujian by temperature conditions culturing area.And
Hainan is located in subtropical zone, torrid areas, and ocean temperature is of a relatively high, the hatching and nursery of suitable egg-shaped pompano.At present, three provinces and regions
All egg-shaped pompanos cultivate seed both from Hainan Region.Due to the phenomenon of egg-shaped pompano generally existing inbreeding, cause
Seed germplasm is degenerated, slow-growing, is increasingly highlighted the problems such as disease breaks out.The man of wild origin that traditional seed breeding industry is used is domestic
Pattern is faced with huge challenge, for egg-shaped pompano carry out fine-variety breeding become for aquaculture researcher it is extremely urgent appoint
Business.
Family selective breeding is the important foundation means of fish breeding, is to be substantially better than its parent according to certain or a few proterties
Category, production performance are significantly higher than being mixed with different types of primitive horde for its relatives and select some defect individuals and reserve seed for planting, and set up several
Individual or several familys simultaneously raise up seed, and by generation compared with initial population and check variety, selecting and remain, those meet original selection
The excellent system of index, and then carry out strain performance measurement.In fish breeding, family selective breeding is particularly important, nearly all to educate
The step for being required to by family selective breeding into species.The foundation of current Technique in Fishes family is generally handed over 1 (hero) using 1 (female)
With production filial generation.The family negligible amounts that such mode is set up, and it is affected by environment larger individually to manage each family, it is right
Families selecting is unfavorable；And individual physical markings are carried out to mixed family, it is extremely difficult in the case of more family.It is right at present
The judgement for breeding maturity and male and female sex in egg-shaped pompano is relatively difficult, also and does not apply to the mode of production of 1 pair 1.Therefore it is right
In the foundation of egg-shaped pompano family and the identification of family be always insoluble problem, the avette silvery pomfret for largely hindering
Scad breeding works.
Microsatellite DNA (Microsatellites DNA) is a kind of moderate being distributed widely in eukaryotic gene group
Repetitive sequence.Microsatellite marker is widely used in population genetic variations point with its rich polymorphism, the advantage of codominant inheritance
The fields such as analysis, character analysis, Germplasm Identification, Parentage determination, genetic mapping.The microsatellite hereditary information of parent can be by breeding
Filial generation is passed to, micro-satellite labeling technique distinguishes individual affiliated family by detecting the individual microsatellite difference of different parent-offsprings.Mesh
It is preceding there is not yet by microsatellite marker be used for egg-shaped pompano Parentage determination report.
The content of the invention
It is avette silvery pomfret it is an object of the invention to provide a kind of method that utilization microsatellite marker identifies egg-shaped pompano family
Scad breedings provide the molecular labeling instrument of paternity test.
To achieve these goals, the technical scheme is that：A kind of side of egg-shaped pompano family paternity test is provided
Method, the method comprises the following steps：
1) selection egg-shaped pompano build is larger, and individual 87 tail, as parent, parent's mixing is carried out the speed of growth faster
Artificial propagation；10,000 tails are randomly selected when fry grows to 10 centimeters and is transferred to cage culture, as the family group for paternity test
Body；
2) selecting step 1) egg-shaped pompano breed parent isozyme, extract genomic DNA, with DNA as template, choosing
Nested PCR amplification is carried out with 51 pairs of micro-satellite primers；
3) step 2) described in nested PCR amplification method refer in PCR system comprising 3 primers：Locus specificity forward direction is drawn
Thing, its 5 ' end adds M13 sequences (5 '-TGTAAAACGACGGCCAGT-3 ')；Locus specificity reverse primer；FAM、PET、
The M13 universal primers of tetra- kinds of fluorescence labelings of VIC, NED；PCR reaction systems 20ul：50ng DNA, 2ul dNTP (2.5mM),
2ul10 × Buffer mixture, 1U rTaq, 0.4ul containing plus universal primer M13 forward primer (10mM), 0.5ul is anti-
To primer, the fluorescence labeling universal primer of 0.1ul, ddH2O is supplied；
4) egg-shaped pompano parent in 51 PCR primers of microsatellite locus through ABI3730 type Genetic Analysers, according to fluorescence
Mark carries out genotyping, using Genemapper3.7 software sunykatuib analyses, filters out and is adapted to identify that egg-shaped pompano raises together with house
Effective microsatellite locus 11 of system：ZD13、ZD15、TBG008、ZD02、ZD03、ZD04、ZD11、ZD12、ZD01、ZD10、
TBG016；
5) 10,000 filial generations of egg-shaped pompano were raised together with into for 7 monthly ages, 2000 tails individuality is randomly selected, in every muscle of back of fish
Implantation electronic marker and clip each individual isozyme, extract genes of individuals group DNA, selecting step 4) in 11 it is effectively micro-
Satellite site, analyzes the genotype of filial generation；
6) different filial generations are entered using the softwares of PAPA 2.0 in 11 genotype of microsatellite locus according to parent and filial generation
Row is distinguished, and identifies the Parent of offspring individual.
Wherein step 4) described in 11 couple effectively micro-satellite primers nucleotide sequence it is as follows.
The egg-shaped pompano paternity test micro-satellite primers of table 1
* four kinds of M13 primers of fluorescence labeling are respectively synthesized, are used cooperatively with egg-shaped pompano primer
The present invention has advantages below：
1. the present invention prepares mixed family by the way of parent fish population breeds, without artificial one-to-one pairing breeding.Can
It is not easily distinguishable for male and female sex and the uppity egg-shaped pompano artificial propagation of maturity and family breeding structure, greatly solution
Determine the problem of this type fish family fine-variety breeding.
2. in the present invention filial generation of mixed family since embryonated egg to this stage of marketable fish all under same environment
Cultivation, can avoid the error caused by independent family breeding environment difference, improve the degree of accuracy of family selective breeding.
3. the present invention need not maintain huge isolation from nursery by the way of family is cultivated in mixing to half adult fish stage
Breeding facility, greatlys save management and aquaculture cost.
4. the present invention uses nested PCR amplification method, from tetra- kinds of fluorescence labelings of FAM, PET, VIC, NED to universal primer
M13 is marked, and using the end of microsatellite forward primer 5 ' plus the method for M13 sequences, during for identifying individual more, can be greatly saved
The expense of primer fluorescence labeling.
Brief description of the drawings
The PCR amplifications of Fig. 1 primers TBG008；
The PCR amplifications of Fig. 2 primers ZD15；
Fig. 3 ZD02 sites allele goes out peak figure；
Fig. 4 ZD03 sites allele goes out peak figure；
Specific embodiment
1) egg-shaped pompano family breed and filial generation cultivation
87 tail defect individuals are selected from egg-shaped pompano natural population (Hainan Region) as parent, is planted in its muscle of back
Enter electronic marker and measure each individual growth traits data.Every fritter fin ray of parent's clip one, is stored in 95% alcohol
In, it is stored in -20 DEG C.Parent carries out artificial induced spawning by injecting the method for hormone, and 87 tail parent populations are injected, and injects the side of hormone
Method：Disposable injection human chorionic gonadtropin (HCG), injection dosage：200IU/kg；Luteotropin releasing hormone d-ala analog
(LHRH-A2), injection dosage：3ug/kg.87 tail parent population common property 10kg embryonated eggs, it is 4 mu to take 1.2kg embryonated eggs and be put into area
Hatched in pond, after the cultivation of the tail of prelarva 1,120,000 being estimated after 1d 60 days, prelarva average length is 10 centimeters, randomly selects 10,000
Bar is transferred to 6m × 6m cage cultures.2200 tails are randomly selected from 10,000 when cultivating for 7 monthly age it is transferred to another 6m × 6m net cage and supports
Grow and squeeze into electronic marker, Taking Pictures recording growth traits and clip each filial generation tail fin tissue are stored in 95% alcohol, are preserved
In -20 DEG C.Parent and the individual genomic DNA of family are extracted using ammonium acetate method.
2) screening of PCR primer and amplification reaction system
Have chosen 51 egg-shaped pompano microsatellite locus enter performing PCR amplification, most site expanding effect it is not good or
Difference is smaller in egg-shaped pompano colony, finally filters out that 11 pairs of genetic polymorphisms are high, and the primer of amplification efficiency stabilization is used for avette
Silvery pomfret Scad paternity tests, 11 pairs of effective microsatellite marker amplimers and condition are as shown in table 1.PCR reaction systems are 20ul：10×
PCR Buffer 2ul, dNTP mixture (2.5mM each) 2ul, TaKaRa Taq 0.2ul (5U/ul) (precious bioengineering
Dalian Co., Ltd produces), the end of 10umol/L forward primers 5 ' adds M13 universal primers 0.4ul, 10umol/L reverse primer
0.5ul, 10umol/L M13 fluorescent primers 0.1ul.Amplified reaction is completed in ABI2720PCR systems, and PCR programs are：94℃
Predegeneration 5min；94 DEG C of denaturation 30s；Tm annealing 30s；72 DEG C of extension 30s, reaction carries out 32 circulations, 94 DEG C of denaturation 30s；53
DEG C annealing 30s；72 DEG C of extension 30s, reaction carries out 8 circulations；It is last to extend 10min at 72 DEG C.Wherein Tm values are different according to table 1
Primer annealing temperature sets.The amplification of Fig. 1, Fig. 2 exposition microsatellite locus.
3) microsatellite locus genotyping
After the completion of PCR reactions, posting to Beijing physico-chemical analysis center carries out machine testing, and detecting instrument used is
ABI3730 Genetic Analysers.Genotyping is done with software Gene Mapper3.7, parent and filial generation microsatellite position are read respectively
Point gene type.Fig. 3, Fig. 4 are the peak figure of moiety site.
4) effectively selection and egg-shaped pompano the paternity test analysis of microsatellite locus
Using step 4) method analyze 87 tail egg-shaped pompano parents and 2200 odd amount in addition to the round numbers generation in 51 pairs of bases of microsatellite locus
Because of type, by the genotype of unknown parent's sex module simulation multiplication family filial generation in software PAPA 2.0 and parent genotype
With relation, screen 11 microsatellite locus (ZD13, ZD15, TBG008, ZD02, ZD03, ZD04, ZD11, ZD12, ZD01,
ZD10, TBG016) paternity test accuracy rate can reach 90.625% (table 2).
5) interpretation of result
96 tails are expanded in 11 microsatellite locus raise together with family progeny to 87 tail egg-shaped pompano parents and 2200 tails
And Genotyping.The softwares of genotype application Gene Mapper 3.7 are analyzed, egg-shaped pompano paternity test application PAPA 2.0
The unknown parent's sex module of software is analyzed.To ensure the accuracy of experimental result, only guaranteed all microsatellite seats are complete
The match is successful in portion, and meets the just confirmation parent child relationship of parent's mating system.It is final to confirm the father and mother that 87 tails raise together with filial generation, parent-offspring
Identification rate is 90.625%.
The tail of 2 egg-shaped pompano of table 96 individuality paternity test information table
Above disclosed is only presently preferred embodiments of the present invention, can not limit the right of the present invention with this certainly
Scope, therefore the equivalent variations made according to the claims in the present invention, still fall within the scope that the present invention is covered.
Claims (1)
1. a kind of method of egg-shaped pompano family paternity test, the method comprises the following steps：
1) selection egg-shaped pompano build is larger, and parent, as parent, is mixed into pedestrian's work to the speed of growth by individual 87 tail faster
Breeding；10,000 tails are randomly selected when fry grows to 10 centimeters and is transferred to cage culture, as the family colony for paternity test；
2) selecting step 1) egg-shaped pompano breed parent isozyme, extract genomic DNA, with DNA as template, from 51
Nested PCR amplification is carried out to micro-satellite primers；
3) step 2) described in nested PCR amplification method refer in PCR system comprising 3 primers：Locus specificity forward primer, its
5 ' ends add the-TGTAAAACGACGGCCAGT-3 ' of M13 sequences 5 '；Locus specificity reverse primer；FAM, PET, VIC, NED tetra-
Plant the M13 universal primers of fluorescence labeling；The μ L of PCR reaction systems 20：50ng DNA, 2 μ L dNTP, 2 10 × Buffer of μ L
Mixture, 1U rTaq, 0.4 μ L containing plus universal primer M13 forward primer 10mM, 0.5 μ L reverse primers, 0.1 μ L's is glimmering
Signal universal primer, ddH2O is supplied；
4) egg-shaped pompano parent in 51 PCR primers of microsatellite locus through ABI3730 type Genetic Analysers, according to fluorescence labeling
Genotyping is carried out, using Genemapper3.7 software sunykatuib analyses, is filtered out and is adapted to identify that egg-shaped pompano raises together with family
Effective microsatellite locus 11：ZD13、ZD15、TBG008、ZD02、ZD03、ZD04、ZD11、ZD12、ZD01、ZD10、
TBG016；Described 11 effective microsatellite locus are to be expanded to obtain by following primer sequence 5 ' -3 '：
ZD13-F：TGTAAAACGACGGCCAGTGGCTAGCTTTGCATTGTGTG；
ZD13-R：CAGCCCAGTCAGTCCCTACT；
ZD15-F：TGTAAAACGACGGCCAGTGACGTGTTCCACAGCAAGAA；
ZD15-R：AGGAATGGTCCCAAAGAATG；
TBG008-F：TGTAAAACGACGGCCAGTTCGCGACAAACTTTAACTCATCTC；
TBG008-R：AGCATTTTCACCTCCTCCATTG；
ZD02-F：TGTAAAACGACGGCCAGTTTTAGGACACCATCCCCTCA；
ZD02-R：GCTCCTGTGGAGGACAGAGA；
ZD03-F：TGTAAAACGACGGCCAGTATATCAGCGTCCACCCAAAC；
ZD03-R：CTTTTCCATCTCTCCCCTCA；
ZD04-F：TGTAAAACGACGGCCAGTGCTTGTGGAGACCATGACG；
ZD04-R：CTCCTGGAGGAACTGTGGAG；
ZD11-F：TGTAAAACGACGGCCAGTAGAGAGCAGGACGTCCAAAA；
ZD11-R：CTTTTCCATCTCTCCCCTCA；
ZD12-F：TGTAAAACGACGGCCAGTCCACCATCAATCAGCTGTCA；
ZD12-R：AGGTGCTCCACAGATGTTCC；
ZD01-F：TGTAAAACGACGGCCAGTTGCTTGAAAAATCAGGCAAG；
ZD01-R：TGCCAGGGAAAAGAGAGAGA；
ZD10-F：TGTAAAACGACGGCCAGTGGTCTGTAGAGAACCAGAACAGTC；
ZD10-R：GCTCCTGTGGAGGACAGAGA；
TBG016-F：TGTAAAACGACGGCCAGTTCTAGAAATACATCCTGGGTCACT；
TBG016-R：GAACCGGAGTCTGTAAACAAGAT；
5) 10,000 filial generations of egg-shaped pompano were raised together with into for 7 monthly ages, randomly selects 2000 tails individuality, in every muscle of back implantation of fish
Electronic marker and clip each individual isozyme, extract genes of individuals group DNA, selecting step 4) in 11 effective microsatellites
Site, analyzes the genotype of filial generation；
6) area is carried out to different filial generations using the softwares of PAPA 2.0 in 11 genotype of microsatellite locus according to parent and filial generation
Point, identify the Parent of offspring individual.
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| CN109457035B (en) * | 2018-11-26 | 2020-09-18 | 中国水产科学研究院南海水产研究所 | SSR fluorescence labeling primer for parent-child identification of trachinotus ovatus and application thereof |
| CN110150190B (en) * | 2019-06-28 | 2021-10-26 | 海南晨海水产有限公司 | Colony breeding method for egg-shaped pompano |
| CN110521637B (en) * | 2019-08-26 | 2021-11-09 | 中国水产科学研究院南海水产研究所 | Method for constructing full-sibling family of egg-shaped pompano |
| CN111088370B (en) * | 2020-01-20 | 2022-06-21 | 中国水产科学研究院南海水产研究所 | Sex-specific molecular marker primer for Trachinotus ovatus, identification method and application of sex-specific molecular marker primer |
| CN117051130B (en) * | 2023-10-11 | 2023-12-22 | 中国水产科学研究院南海水产研究所 | SNP molecular marker associated with streptococcus agalactiae resistance of trachinotus ovatus and application thereof |
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