CN105603097A - Microsatellite marker primers used for pinctada fucata martensii microsatellite family identification, identification method and application - Google Patents
Microsatellite marker primers used for pinctada fucata martensii microsatellite family identification, identification method and application Download PDFInfo
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Abstract
The invention discloses microsatellite marker primers used for pinctada fucata martensii microsatellite family identification. The microsatellite marker primers include nine primer pairs in total, and the nine primer pairs are PF-3, PF-4, PF-11, PF-13, PF-16, PF-27, PF-30, PF-44 and PF-48 respectively. The invention further discloses an identification method for the pinctada fucata martensii microsatellite family and application of the microsatellite marker primers to pinctada fucata martensii microsatellite family identification. By the adoption of the microsatellite marker primers, a paternity test platform is set up on pinctada fucata martensii with polymorphic microsatellite markers marked by fluorescence for the first time, and the identification accuracy reaches 90.24%; different families and sources of the pinctada fucata martensii can be effectively and fast identified, and a basis is provided for breeding, reproduction matching and enhancement and releasing assessment of the pinctada fucata martensii.
Description
Technical field
The invention belongs to the molecular marking technique field of shellfish genetic breeding, be specifically related to a kind of for Hepu pearl oysterThe microsatellite marker primer of the micro-satellite Parentage determination of shellfish and authentication method and application.
Background technology
Pinctada fucata (Pinctadafucata) also claims " pteria martensii ", Bivalvia, and Pteriidae, adheres toLive at the bottom of water quality clarification has the shallow sea of coral reef or rocky gravel, be distributed in the South Sea, Japan also produces, except producing pearlOutward, meat is edible, and shell can button processed, is one of China and sea water pearls is cultivated in the world main species, ChinaThe South China coastal pearl that pinctada fucata produces is " Nan Zhu " world-famous.
Owing to not focusing on seed selection kind in long-term breeding process, and aquaculture model is outmoded, and cultivation place is aging,Grow cultured pearls backward in technique, cause cultivation pinctada fucata poor growth, nacre secretion capacity declines, the ability of growing cultured pearlsReduce, cause cultured pearl Quality and yield all obviously to decline, bead is little, and pearl layer is thin, shade deviation, treasurePearl culture benefit is low, and many raisers abandon cultured pearl and change foster other kind, have seriously limited " Nan Zhu "The development of aquaculture industry. Therefore, carry out as early as possible the work of pinctada fucata fine-variety breeding be promote its aquaculture stable,The necessary means developing in a healthy way.
In shellfish genetic breeding research, pedigree information extremely closes for the seed selection of family and parent's management clearlyImportant, be also affiliation between clear and definite individuality simultaneously, set up the important means of correct pedigree. Pass through affiliationQualification, carries out pedigree analysis research and can illustrate breeding success rate, many relevant something lost such as enhancement releasing young diffusionPass the problem in science that aspect and breeding ecology are learned. In pinctada fucata breeding process, parent's quantity is notCan be a lot, by for artificial propagation, caused a lot of seeds to occur decay in addition.
Therefore the breeding that effectively pedigree qualification can be understood breeding parent is worth, simultaneously to improve breeding efficiency andProduction practices are all significant. That current aquatic livestock is taking micro-satellite somatotype as basic paternity test technologySpectrum is one of most widely used general the most reliable means in confirming. Scallop, Pacific oyster, ear Bao, all are applied toReceive in the aquatic products economic animal breedings such as shore prawn, Chinese prawn, lefteye flounder.
At present, very few about the report that microsatellite marker is applied to Pinctada fucata family identification research.
Summary of the invention
First technical problem to be solved by this invention is to provide a kind of for the micro-satellite family of pinctada fucataThe microsatellite marker primer of qualification.
Second technical problem to be solved by this invention is to provide one and utilizes above-mentioned microsatellite marker primer to enterThe authentication method of the micro-satellite family of row pinctada fucata, the method is easy to somatotype simultaneously and detects, can be for HepuThe population genetic structure of nacre, genetic thremmatology assessment and paternity test etc., can significantly save experiment simultaneouslyCost.
Last technical problem to be solved by this invention is to provide one and utilizes above-mentioned for pinctada fucataThe application of the microsatellite marker primer of micro-satellite Parentage determination in Pinctada fucata family qualification.
First technical problem to be solved by this invention is achieved through the following technical solutions: one is used forThe microsatellite marker primer of the micro-satellite Parentage determination of pinctada fucata, described microsatellite marker primer has 9 and drawsThing pair, is respectively PF-3, PF-4, PF-11, PF-13, PF-16, PF-27, PF-30, PF-44 and PF-48,Wherein:
The nucleotide sequence of primer pair PF-3 is as shown in SEQIDNO:1 and SEQIDNO:2;
The nucleotide sequence of primer pair PF-4 is as shown in SEQIDNO:3 and SEQIDNO:4;
The nucleotide sequence of primer pair PF-11 is as shown in SEQIDNO:5 and SEQIDNO:6;
The nucleotide sequence of primer pair PF-13 is as shown in SEQIDNO:7 and SEQIDNO:8;
The nucleotide sequence of primer pair PF-16 is as shown in SEQIDNO:9 and SEQIDNO:10;
The nucleotide sequence of primer pair PF-27 is as shown in SEQIDNO:11 and SEQIDNO:12;
The nucleotide sequence of primer pair PF-30 is as shown in SEQIDNO:13 and SEQIDNO:14;
The nucleotide sequence of primer pair PF-44 is as shown in SEQIDNO:15 and SEQIDNO:16;
The nucleotide sequence of primer pair PF-48 is as shown in SEQIDNO:17 and SEQIDNO:18.
Second technical problem to be solved by this invention is achieved through the following technical solutions: above-mentioned HepuThe authentication method of the micro-satellite family of nacre, comprises the following steps:
(1) pinctada fucata parent and daughter DNA extract: choose pinctada fucata and carry out entirely as parentThe breeding of compatriot's family, chooses parent and filial generation musculature after breeding, adopt Magen animal DNA to extract examinationAgent box extracts, and the DNA that obtains pinctada fucata parent and filial generation is stand-by as template;
(2) microsatellite marker primer fluorescent decoration: choose 9 primer pairs in claim 1, be divided into 3Group, wherein PF-4, PF-11 and PF-30 are one group, PF-13, PF-27 and PF-44 are one group, PF-3,PF-16 and PF-48 are one group, use respectively FAM, HEX and ROX at the forward primer 5 ' end of every group of primerThree kinds of different fluorophors are modified, for pcr analysis;
(3) fluorescent PCR: adopt Fluorescence PCR to utilize 9 of the middle fluorophor modified of step (2)Individual primer pair carries out pcr amplification to the DNA of the parent in step (1) and filial generation respectively, and amplified production is pressedMethod according to grouping in step (2) is mixed, and mixture is as upper machine testing sample;
(4) the micro-satellite Parentage determination of pinctada fucata: adopt genetic analysis instrument to divide upper machine testing sampleAnalyse, read the genotype of each sample, parent genotype and filial generation genotype are analyzed, judge filial generationThe Parent of body, micro-satellite family of acquisition pinctada fucata.
In the authentication method of the micro-satellite family of above-mentioned pinctada fucata:
When the middle Fluorescence PCR of step (3), 20 μ L reaction systems preferably include 15.2 μ L distilled waters, 2 μ L10 × PCR buffer solution, concentration is the each 0.6 μ L of the positive and negative primer of 10 μ m/L, concentration is 10mm/LDNTP0.3 μ L, concentration is the Taq enzyme 0.3 μ L of 5U/ μ L, 1 μ LDNA template.
When the middle Fluorescence PCR of step (3), preferably 95 DEG C of denaturation 5min, 95 DEG C of sex change 30s, 56 DEG CAnnealing 30s, 72 DEG C are extended 30s, and 30 circulations are carried out in reaction, and last 72 DEG C are extended 10min again.
In step (4), preferably adopt software CERVUS3.0 software to enter parent genotype and filial generation genotypeRow is analyzed, and judges the Parent of offspring individual.
Adopt software CERVUS3.0 can calculate parent and filial generation on each micro-satellite seat gene frequency,Heterozygosity, expectation heterozygosity, polymorphism information content, average probability of exclusion, Hardy-Weinberg balance and nothingThe information such as effect gene frequency, thereby the Parent of judgement offspring individual.
Further, the authentication method of the micro-satellite family of a kind of pinctada fucata provided by the invention, comprises followingStep:
(1) pinctada fucata DNA extracts
Choose the good pinctada fucata of healthy development and carry out family full-sibs breeding as parent, clip parent andFilial generation closed shell muscular tissue sample is put in absolute ethyl alcohol, adopts Magen mollusk DNA to extract kitOperation, detects after quality and concentration, is diluted to 100ng/ μ L, deposits in-20 DEG C and saves backup;
(2) screening of polymorphic micro-satellite markers primer and primer are synthetic
Choose 9 pairs of micro-satellite primers, be divided into 3 groups according to clip size, wherein, primer PF-4, PF-11,PF-30, is one group, primer PF-13, and PF-27, PF-44, is one group, primer PF-3, PF-16, PF-48,It is one group; Use respectively FAM at forward primer 5 ' end, HEX, tri-kinds of different fluorophors of ROX are repaiiedDecorations, for pcr analysis;
(3) fluorescent PCR
Adopt Fluorescence PCR parent and daughter DNA sample are carried out to pcr amplification, amplified production according toIn step (2), the method for selected micro-satellite primers grouping is mixed, as the sample of upper machine testing;
(3) microsatellite locus Genotyping and the micro-satellite Parentage determination of spot joint pinctada fucata
Sample carries out somatotype on ABI3730XL genetic analysis instrument,, uses as internal reference with GS-500GeneMapperV3.2 software reads the genotype of each sample, adopts CERVUS3.0 software to parent's baseBecause type and filial generation genotype are analyzed, judge the Parent of offspring individual.
The 3rd technical problem to be solved by this invention is achieved through the following technical solutions: above-mentioned forThe application of the microsatellite marker primer of the micro-satellite Parentage determination of pinctada fucata in Pinctada fucata family qualification.
Compared with prior art, tool of the present invention has the following advantages:
(1) adopt the primer in the present invention, on pinctada fucata, utilize first fluorescently-labeled polymorphic micro-defendingAsterisk note has been set up paternity test platform, and its qualification rate of accuracy reached is to 90.24%;
(2) the inventive method can be differentiated the different familys of pinctada fucata and source quickly and efficiently, is HepuThe seed selection of nacre, breeding combo and enhancement releasing assessment provide foundation;
(3) the present invention is according to combine different with fluorescently-labeled color of micro-satellite primers amplified fragments size,By 3 micro-satellite primers respectively the DNA product after pcr amplification mix sample detection, than commonPCR detection method has improved 3 times of efficiency, has saved cost and time simultaneously;
(4) the microsatellite locus allele number that the present invention selects is more, and polymorphism is high, can be for closingThe population genetic structure of Pu nacre, genetic thremmatology assessment and paternity test etc., can significantly save reality simultaneouslyTest cost.
Detailed description of the invention
Below in conjunction with specific implementation method, the present invention is described in detail.
Embodiment 1 for the screening of the microsatellite marker primer of the micro-satellite Parentage determination of pinctada fucata with synthetic
Screen by transcribing group order-checking the unigene that contains the micro-satellite repetitive sequence of pinctada fucata, then fromPinctada fucata is transcribed group library and will the micro-satellite specificity of the sequences Design that contains microsatellite locus wherein be detectedPrimer, detects its polymorphism, through finally having selected 9 pairs of bands clear after screening, and the primer that polymorphism is highAs the primer of Parentage determination, primer is synthetic in Shanghai Sheng Gong bioengineering limited company.
Between the suitable and mark in microsatellite marker primer rich polymorphism, the clip size interval selected in the present invention notChain, be easy to somatotype simultaneously and detect, can be for the population genetic structure of pinctada fucata, genetic thremmatology is commentedEstimate and paternity test etc., can significantly save experimental cost simultaneously.
Table 1 is for the micro-satellite primers information of Pinctada fucata family qualification
The authentication method of the micro-satellite family of embodiment 2 pinctada fucata
(1) pinctada fucata parent and daughter DNA extract
(1) foundation of Pinctada fucata family
Choose in four kinds of shell chromaticity systems of pinctada fucata and 1 fast-growth strain and select pinctada fucata male and femaleEach 12, carry out artificial propagation, male and female proportioning 1:1, sets up 12 family full-sibses. The each family of clipParent's closed shell muscular tissue is put in absolute ethyl alcohol, and carries out family information recording, in-20 DEG C of preservations. By 12Individual family is placed on respectively different cage tools and raises, and after treating half a year, chooses at random 14 shellfishes from each family,Get its closed shell flesh absolute ethyl alcohol and fix, as the sample of Parentage determination.
(2) extraction of pinctada fucata parent and progeny genome DNA
Adopt Magen mollusk tissue DNA to extract kit, 20-50mg tissue sample is processed intoThe little fragment of trying one's best, and be transferred in 1.5mL centrifuge tube. Add 550 μ LBufferMTL and 20 μ L eggsWhite enzyme K, vortex mixes. 55 DEG C of vibrate incubation 3h or digested overnight samples. Add 5 μ LRNaseSolutionTo digestive juice, put upside down and mix. Room temperature leaves standstill 30-60min digestion RNA. The centrifugal 3min of 13000xg.Shift supernatant to new 2.0mL centrifuge tube. Add 500 μ LBufferDL to digestive juice, vortexMix 20s, 70 DEG C of water-bath 10min. Add 500 μ L absolute ethyl alcohols to digestive juice, vortex mixes 20s.HipuregDNAMiniColumn is contained in 2mL collecting pipe. Shift mixed liquor to pillar,The centrifugal 1min of 10000xg. Pour out efflux, pillar is reinstalled in collecting pipe, add 500 μ LBufferGW1 to pillar, the centrifugal 1min of 10000xg. Pour out efflux, pillar be reinstalled in collecting pipe,Add 650 μ LBufferGW2 to pillar, the centrifugal 1min of 10000xg. Pour out efflux, pillar weightNew clothes reclaim in collector, the centrifugal 2min of 10000xg. Pillar is contained in new 1.5mL centrifuge tube. Add30-200 μ L is preheated to the film central authorities of 55 DEG C of BufferAE to pillar. Place 2min, 10000xg centrifugal 1Min. Abandon DNA column, by NanoDropND-1000 UV spectrophotometer measuring DNA concentrationAnd quality, by each DNA Sample Dilution to 100ng/ μ L, DNA is preserved-20 DEG C for subsequent use.
(2) microsatellite marker primer fluorescent decoration
Choose 9 primer pairs in embodiment 1, be divided into 3 groups, wherein PF-4, PF-11 and PF-30 are oneGroup, PF-13, PF-27 and PF-44 are one group, PF-3, PF-16 and PF-48 are one group, at every group of primerForward primer 5 ' end modify with FAM, HEX and tri-kinds of different fluorophors of ROX respectively, toolBody is as shown in table 1, for pcr analysis;
Adopt Fluorescence PCR to utilize 9 primer pairs of above-mentioned fluorophor modified respectively to above-mentioned parentAnd the DNA of filial generation carries out pcr amplification, amplified production mixes according to the method for above-mentioned grouping, mixtureAs upper machine testing sample.
PCR reaction system is 20 μ L: contain 15.2 μ L distilled waters, and 2 μ L10 × PCR buffer solutions, positive and negativeThe each 0.6 μ L of primer (10 μ m/L), dNTP0.3 μ L (10mm/L), 0.3 μ LTaq enzyme (5U/ μ L),1 μ LDNA template.
Amplified reaction PCR program is 95 DEG C of denaturation 5min; 95 DEG C of sex change 30s, 56 DEG C of annealing 30s,72 DEG C are extended 30s, and 30 circulations are carried out in reaction; Last 72 DEG C are extended 10min again.
(3) microsatellite locus Genotyping and Parentage determination
(1) microsatellite locus Genotyping
Amplified production carries out somatotype on ABI3730XL genetic analysis instrument, uses GS-500LIZ as internal reference,Read individual genotype with GeneMapperV3.2 software.
Adopt software CERVUS3.0 to calculate parent and filial generation gene frequency, assorted on each micro-satellite seatRight, expect heterozygosity, polymorphism information content, average probability of exclusion, Hardy-Weinberg balance and invalidGene frequency (in table 2).
Genetic diversity statistics and the probability of exclusion of a table 29 microsatellite locus primer
Note: k is allele number, and Ho is for observing heterozygosity, HEFor expecting heterozygosity, PIC is polymorphic content, Excl1Elimination factor during for parents' the unknown, elimination factor when Excl2 is known single parent, HW is hardy weinberg equilibrium inspection, ND representsCheck, * * * represents to depart from extremely remarkable, and NS represents to depart from not remarkable, and F (Null) represents amorph frequency.
(2) Parentage determination result
In sunykatuib analysis, (use CERVUS3.0), simulate and produce 10000 filial generations with 12 couples of parents,In 85% and 90% fiducial interval range, paternity test success rate all can reach 97%.
In 164 individualities of 12 familys of reality qualification, there are 16 examples not find real Parent. FromIn candidate parent, finding real parental probability is 90.24%, can meet in genetic breeding pedigree analysis andThe requirement of enhancement releasing assessment.
Obviously, foregoing is for feature of the present invention is described, and is not limitation of the present invention, relevantThe variation that the those of ordinary skill of technical field is made in corresponding technical field according to the present invention should belong to thisBright protection category.
Claims (6)
1. for a microsatellite marker primer for the micro-satellite Parentage determination of pinctada fucata, it is characterized in that: instituteState microsatellite marker primer and have 9 primer pairs, be respectively PF-3, PF-4, PF-11, PF-13, PF-16,PF-27, PF-30, PF-44 and PF-48, wherein:
The nucleotide sequence of primer pair PF-3 is as shown in SEQIDNO:1 and SEQIDNO:2;
The nucleotide sequence of primer pair PF-4 is as shown in SEQIDNO:3 and SEQIDNO:4;
The nucleotide sequence of primer pair PF-11 is as shown in SEQIDNO:5 and SEQIDNO:6;
The nucleotide sequence of primer pair PF-13 is as shown in SEQIDNO:7 and SEQIDNO:8;
The nucleotide sequence of primer pair PF-16 is as shown in SEQIDNO:9 and SEQIDNO:10;
The nucleotide sequence of primer pair PF-27 is as shown in SEQIDNO:11 and SEQIDNO:12;
The nucleotide sequence of primer pair PF-30 is as shown in SEQIDNO:13 and SEQIDNO:14;
The nucleotide sequence of primer pair PF-44 is as shown in SEQIDNO:15 and SEQIDNO:16;
The nucleotide sequence of primer pair PF-48 is as shown in SEQIDNO:17 and SEQIDNO:18.
2. an authentication method for the micro-satellite family of pinctada fucata, is characterized in that comprising the following steps:
(1) pinctada fucata parent and daughter DNA extract: choose pinctada fucata and carry out entirely as parentThe breeding of compatriot's family, chooses parent and filial generation musculature after breeding, adopt Magen animal DNA to extract examinationAgent box extracts, and the DNA that obtains pinctada fucata parent and filial generation is stand-by as template;
(2) microsatellite marker primer fluorescent decoration: choose 9 primer pairs in claim 1, be divided into 3Group, wherein PF-4, PF-11 and PF-30 are one group, PF-13, PF-27 and PF-44 are one group, PF-3,PF-16 and PF-48 are one group, use respectively FAM, HEX and ROX at the forward primer 5 ' end of every group of primerThree kinds of different fluorophors are modified, for pcr analysis;
(3) fluorescent PCR: adopt Fluorescence PCR to utilize 9 of the middle fluorophor modified of step (2)Individual primer pair carries out pcr amplification to the DNA of the parent in step (1) and filial generation respectively, and amplified production is pressedMethod according to grouping in step (2) is mixed, and mixture is as upper machine testing sample;
(4) the micro-satellite Parentage determination of pinctada fucata: adopt genetic analysis instrument to divide upper machine testing sampleAnalyse, read the genotype of each sample, parent genotype and filial generation genotype are analyzed, judge filial generationThe Parent of body, micro-satellite family of acquisition pinctada fucata.
3. the authentication method of the micro-satellite family of pinctada fucata according to claim 2, is characterized in that:When the middle Fluorescence PCR of step (3), 20 μ L reaction systems comprise 15.2 μ L distilled waters, 2 μ L10 × PCRBuffer solution, concentration is the each 0.6 μ L of the positive and negative primer of 10 μ m/L, the dNTP0.3 μ L that concentration is 10mm/L,Concentration is the Taq enzyme 0.3 μ L of 5U/ μ L, 1 μ LDNA template.
4. the authentication method of the micro-satellite family of pinctada fucata according to claim 2, is characterized in that:When the middle Fluorescence PCR of step (3), 95 DEG C of denaturation 5min, 95 DEG C of sex change 30s, 56 DEG C of annealing30s, 72 DEG C are extended 30s, and 30 circulations are carried out in reaction, and last 72 DEG C are extended 10min again.
5. the authentication method of the micro-satellite family of pinctada fucata according to claim 2, is characterized in that:In step (4), adopt software CERVUS3.0 software to analyze parent genotype and filial generation genotype,Judge the Parent of offspring individual.
6. the microsatellite marker primer for the micro-satellite Parentage determination of pinctada fucata claimed in claim 1 existsApplication in Pinctada fucata family qualification.
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| CN110218800A (en) * | 2019-07-10 | 2019-09-10 | 广东海洋大学 | One kind SNP marker relevant to pteria martensii growth traits and its application |
| CN110295238A (en) * | 2019-07-10 | 2019-10-01 | 广东海洋大学 | A kind of relevant OSR1 gene SNP molecular labeling of pteria martensii growth traits and its application |
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