CN107385094A - A kind of multiple PCR primer, method and application for Environment of Litopenaeus vannamei Low germplasm identification - Google Patents
A kind of multiple PCR primer, method and application for Environment of Litopenaeus vannamei Low germplasm identification Download PDFInfo
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- CN107385094A CN107385094A CN201710803979.XA CN201710803979A CN107385094A CN 107385094 A CN107385094 A CN 107385094A CN 201710803979 A CN201710803979 A CN 201710803979A CN 107385094 A CN107385094 A CN 107385094A
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Abstract
The invention provides a kind of multiple PCR primer, method and application for Environment of Litopenaeus vannamei Low germplasm identification.The present invention is by extracting Environment of Litopenaeus vannamei Low genome, utilize tri- microsatellite DNA core sequences of Environment of Litopenaeus vannamei Low LvL1, LvL2, LvL3, design specific primer and add fluorescence labeling FAM, HEX and TAMRA fluorescence labeling respectively, utilize multiple PCR technique, simultaneously three primer pair Environment of Litopenaeus vannamei Low genomic DNAs are entered with performing PCR amplification, after sequencing, the genotype of each individual is determined according to DNA fragmentation size.Present invention is mainly applied to Environment of Litopenaeus vannamei Low germ plasm resource and analysis of genetic diversity, Parentage determination, pedigree is built, molecular population genetics, the structure of genetic map, the positioning of important economical trait and the research of functional gene.
Description
Technical field
The present invention relates to molecular biology, genetic breeding field, and in particular to one kind is used for Environment of Litopenaeus vannamei Low
The multiple PCR primer, method and application of (Litopenaeus vannamei) germplasm identification.More particularly, to one kind
For tri- microsatellite DNA mark detections of Penaeus Environment of Litopenaeus vannamei Low LvL1, LvL2, LvL3 and Penaeus Environment of Litopenaeus vannamei Low germplasm
Multiple PCR primer, method and the application of resource identification.
Background technology
Environment of Litopenaeus vannamei Low (Litopenaeus vannamei) is commonly called as Penaeus Vannmei (Penaeus vannamei), classification
It is under the jurisdiction of Arthropoda (Arthropoda), Crustachia (Crustacea), Decapoda (Decapoda), Penaeidae on
(Penaeidae), Penaeus (Penaeus), it is a kind of wide temperature, the eurysalinity torrid zone shrimps for originating in Central and South America.Vannamei boone
Prawn is global maximum prawn culturing kind, accounts for more than the 80% of world's prawn culturing total output, and China's cultured output
Highest prawn kind, coastal area of the cultivation scope from Liaoning to the South Sea, the fresh water culture area such as including Hunan.Cause
This, Environment of Litopenaeus vannamei Low occupies critical role in China prawn or even whole world shrimp culture industry.
From 1997 since China's large-scale farming, area gradually expands Environment of Litopenaeus vannamei Low, 2016, China's vannamei boone
Prawn culturing annual production is more than 1,350,000 tons.However, the growth that main foster kind Environment of Litopenaeus vannamei Low occurs in breeding process is delayed
Slowly, the speed of growth is uneven and the problems such as disease frequently breaks out, and main cause is that prawn germ plasm resource is single and variety deterioration.
Prawn kind Geological Problems have had a strong impact on the development of shrimp culture industry, because Environment of Litopenaeus vannamei Low is not belonging to China's native species, I
State does not have an abundant Wild ornamental resources of genetic diversity, improved seeds heavy dependence import, and China needs parent every year according to statistics
Shrimp import volume is up to 300,000 to (200 yuan/to), it is easy to the predicament under one's control of China's prawn culturing industry occurs.Received in face of all
Shore prawn does not have wild resource, the circumstances of existing cultured prawn genetic diversity deficiency, develops new prawn breeding technique and choosing
Educate scheme and just be badly in need of compeling highly necessary to solve the problems, such as in Guangdong or even national prawn scientific and technical personnel as pendulum.
At present, China's fishery genetic breeding improvement work passes through colony or family mainly based on colony or family selective breeding
Between hybridization can realize the enrichment of merit, cultivate the kind of suitable specific aquaculture model.But pass through colony or family
Hybridize the offspring obtained, affiliation is indefinite between its family, individual, and inbreeding and heredity are inevitably resulted in Breeding Process
Multifarious loss.Therefore, it is how easy and effectively identify different groups or family, understand that the heredity between hybridization objects is believed
The distance of breath and affiliation is just particularly important to prawn seed selection.
Multiple PCR technique refers in a PCR reaction system while carries out the amplification of multiple purpose fragments, and selection is multiple
Microsatellite locus establishes multiplexed PCR amplification technology, can carry out parting to multiple microsatellite locus simultaneously, greatly reduce PCR
Cost is expanded, adds the Discussing Convenience of germplasm identification.China Patent Publication No. CN103468796B (denominations of invention：Carry out
The multiple microsatellite identification system of Environment of Litopenaeus vannamei Low Parentage determination and application) disclose a kind of progress Environment of Litopenaeus vannamei Low Parentage determination
Multiple microsatellite identification system and application, method include from Environment of Litopenaeus vannamei Low genome selection different linkage groups have compared with
27 sites of high polymorphism, using two disclosed in the patent multiple microsatellite systems to candidate's Parents of 9 familys and
Filial generation carries out microsatellite parting, and Parentage determination analysis is carried out finally by Cervus softwares.
EST (EST) is the expression fragment of functional gene, the microsatellite locus found wherein can directly with
Functional gene is related, and may be associated with the production traits.Polymorphism information amount (PIC) is that a genetic marker polymorphism can carry
The measurement of the information content of confession, more hereditary information can be obtained using the genetic marker of high PIC values.Therefore, it is necessary to provide one
Genetic marker of the kind available for the high PIC values of Environment of Litopenaeus vannamei Low Parentage determination.
The content of the invention
To draw to solve the above problems, the invention provides a kind of multiplex PCR for Environment of Litopenaeus vannamei Low germplasm identification
Thing, method and application.
The present invention uses bioinformatics method, searches for microsatellite sequence from Environment of Litopenaeus vannamei Low est database, filters out
3 have the microsatellite locus of high polymorphism and design primer, and wherein LvL1 PIC is up to 0.8156, LvL2 0.6327,
LvL3 is 0.7617.The present invention, which is compared to existing report, has following significant advantage：1st, only need to draw using 3 pairs of high polymorphisms
Thing can realizes that to Environment of Litopenaeus vannamei Low germplasm identification and pedigree analysis embodying the present invention has the characteristics of high efficiency；
2nd, 3 pairs of micro-satellite primers are used only in the present invention, reduce the cost of primer synthesis and amplification system, and embodying the present invention has warp
The characteristics of Ji property；3rd, 3 pairs of micro-satellite primers are used only in the present invention, shorten the subsequent data analysis cycle, embody tool of the present invention
There is the characteristics of simplicity.
First aspect present invention provides a kind of multiple PCR primer for Environment of Litopenaeus vannamei Low germplasm identification, described
Multiple PCR primer includes the primer sets of following three pairs of primers composition：
Pair of primers：Sense primer LvL1-F, anti-sense primer LvL1-R；
Second pair of primer：Sense primer LvL2-F, anti-sense primer LvL2-R；
3rd pair of primer：Sense primer LvL3-F, anti-sense primer LvL3-R；
Wherein, sense primer LvL1-F nucleotide sequence includes the nucleotide sequence or and SEQ shown in SEQ ID NO.1
Nucleotide sequence complementary ID NO.1；
Anti-sense primer LvL1-R nucleotide sequence include SEQ ID NO.2 shown in nucleotide sequence or with SEQ ID
Nucleotide sequence complementary NO.2；
Sense primer LvL2-F nucleotide sequence include SEQ ID NO.3 shown in nucleotide sequence or with SEQ ID
Nucleotide sequence complementary NO.3；
Anti-sense primer LvL2-R nucleotide sequence include SEQ ID NO.4 shown in nucleotide sequence or with SEQ ID
Nucleotide sequence complementary NO.4；
Sense primer LvL3-F nucleotide sequence include SEQ ID NO.5 shown in nucleotide sequence or with SEQ ID
Nucleotide sequence complementary NO.5；
Anti-sense primer LvL3-R nucleotide sequence include SEQ ID NO.6 shown in nucleotide sequence or with SEQ ID
Nucleotide sequence complementary NO.6.
Specifically, in the embodiment of the present invention, the nucleotide sequence shown in SEQ ID NO.1-6 is as follows：
1)LvL1-F:AAGGCATTGGCAATCAGCTGAAGTA(SEQ ID NO.1)；
2)LvL1-R:ACGCACACGTACACTTGTAGACAGA(SEQ ID NO.2)；
3)LvL2-F:CGCCCTCTCTCTATTTCTCTCCACCC(SEQ ID NO.3)；
4)LvL2-R:AGAGGGAGCGGAAAGGGGATACAGT(SEQ ID NO.4)；
5)LvL3-F:TGGGTAGTACGCTATATTTGGGAAGC(SEQ ID NO.5)；
6)LvL3-R:ACAGAGAGAAGGAGGGAGTCAGGCA(SEQ ID NO.6)。
In the embodiment of the present invention, " complementation " refers to nucleic acid with another nucleotide sequence by means of traditional Watson-Crick
Base pairing or other non-traditional types form one or more hydrogen bonds.In the embodiment of the present invention, " complementation " has different mutual
Mend percentage, including " complete complementary " or " being substantially complementary ".
" complementing percentage " represent can be formed with second nucleotide sequence in a nucleic acid molecules hydrogen bond (for example, Watson-
Crick base pairing) residue percentage (for example, have among 10 5,6,7,8,9,10 be 50%, 60%,
70%th, 80%, 90% and 100% is complementary)." complete complementary " represents all consecutive residues of a nucleotide sequence and one the
Equal number of consecutive residue in two nucleotide sequences forms hydrogen bond." it is substantially complementary " and refers in a tool as used herein
There are 8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,30,35,40,45,50 or more
On the region of individual nucleotides be at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,
Or 100% complementarity.
In one embodiment of the present invention, what first party of the present invention provided is used for the more of Environment of Litopenaeus vannamei Low germplasm identification
Weight PCR primer includes the primer sets of following three pairs of primers composition：
Pair of primers：Sense primer LvL1-F, anti-sense primer LvL1-R；
Second pair of primer：Sense primer LvL2-F, anti-sense primer LvL2-R；
3rd pair of primer：Sense primer LvL3-F, anti-sense primer LvL3-R；
Wherein, sense primer LvL1-F nucleotides sequence is classified as nucleotide sequence or and SEQ shown in SEQ ID NO.1
Nucleotide sequence complementary ID NO.1；
Anti-sense primer LvL1-R nucleotides sequence be classified as nucleotide sequence shown in SEQ ID NO.2 or with SEQ ID
Nucleotide sequence complementary NO.2；
Sense primer LvL2-F nucleotides sequence be classified as nucleotide sequence shown in SEQ ID NO.3 or with SEQ ID
Nucleotide sequence complementary NO.3；
Anti-sense primer LvL2-R nucleotides sequence be classified as nucleotide sequence shown in SEQ ID NO.4 or with SEQ ID
Nucleotide sequence complementary NO.4；
Sense primer LvL3-F nucleotides sequence be classified as nucleotide sequence shown in SEQ ID NO.5 or with SEQ ID
Nucleotide sequence complementary NO.5；
Anti-sense primer LvL3-R nucleotides sequence be classified as nucleotide sequence shown in SEQ ID NO.6 or with SEQ ID
Nucleotide sequence complementary NO.6.
Second aspect of the present invention provides a kind of multiplex PCR system for Environment of Litopenaeus vannamei Low germplasm identification, including
Multiple PCR primer described in first aspect.
In one embodiment of the invention, in the multiplex PCR system, the multiple PCR primer described in used first aspect
5 terminal modified have fluorescent tag molecule.
It will be appreciated by persons skilled in the art that fluorescent tag molecule can arbitrarily, independently selected from including but unlimited
In following fluorescent tag molecule：FAM (Fluoresceincarboxylic acid), ROX (carboxyl X rhodamines), HEX (chlordene fluorescein), TAMRA (carboxylics
Base tetramethylrhodamine), and the fluorescent tag molecule that each pair of primer pair uses is different.
In one embodiment of the present invention, 5 ends of primer pair LvL1-F, LvL1-R are modified with FAM；Primer pair LvL2-
F, LvL2-R 5 ends are modified with HEX, 5 ends of primer pair LvL3-F, LvL3-R are modified with TAM.
In one embodiment of the invention, in the multiplex PCR system, multiple PCR primer group uses when carrying out multiplex PCR
60 DEG C annealing.
In one embodiment of the invention, in the multiplex PCR system, multiple PCR primer group when carrying out multiplex PCR, it is each just
Reverse primer equimolar is than configuration.
Third aspect present invention provides a kind of method for Environment of Litopenaeus vannamei Low germplasm identification, including：1) obtain
Environment of Litopenaeus vannamei Low genome；2) Environment of Litopenaeus vannamei Low genome is obtained using the multiple PCR primer amplification described in first aspect；3) it is right
Multiplexed PCR amplification product carries out high-flux sequence；4) bioinformatic analysis is carried out to sequencing result, obtains Environment of Litopenaeus vannamei Low kind
Matter resource qualification result.
In one embodiment of the invention, in the step 2), multiplex PCR system reaction solution uses TaKaRa TaqTM Hot
Start Version, are configured to：
0.3 μ l TaKaRa Taq HS (5U/ μ l), 6 μ 10 × PCR of l Buffer, 4.8 μ l dNTP Mixture, three kinds
The forward and reverse primer in SSR sites respectively adds 0.4 μM, DNA profiling 100ng, is supplied with sterile purified water to 40 μ l.
In one embodiment of the invention, in the step 2), multiplexed PCR amplification program is：94 DEG C of pre-degeneration 4min, 35 are followed
Ring：94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extensions 30s, last 72 DEG C of extensions 5min.
In one embodiment of the invention, in the step 2), in the multiplex PCR system, multiple PCR primer group is each forward and reverse
Primer equimolar is than configuration.
Fourth aspect present invention provides a kind of kit for Environment of Litopenaeus vannamei Low germplasm identification, includes first party
The multiple PCR primer that face provides, in addition to multiplexed PCR amplification reagent.
In an embodiment of the present invention, the kit includes conventional supporting reaction reagent and/or consersion unit.Example
Such as, kit can provide one or more reaction or storage buffer solutions.By the available form in specific measure or it can press
The form (such as by concentration or lyophilized form) for adding one or more other components is needed to provide reagent before the use.Buffering
Liquid can be any buffer solution, including but not limited to sodium carbonate buffer, sodium bicarbonate buffer liquid, borate buffer solution, Tris
Buffer solution, MOPS buffer solutions, HEPES buffer solution and combinations thereof.In certain embodiments, the buffer solution is alkaline.At some
In embodiment, the buffer solution has the pH from about 7 to about 10.
Each component in kit of the present invention can provide either individually or in combination, and can be provided in any
In suitable container, such as bottle, bottle, pipe or cardboard.
In an embodiment of the present invention, the kit that the fourth aspect provides also includes：It is endonuclease reaction reagent, double glimmering
One or more reagents in light element enzyme detection reagent.
Fifth aspect present invention provide a kind of multiple PCR primer as described in relation to the first aspect, second aspect provide it is multiple
The kit that PCR system, the method for third aspect offer, the method for fourth aspect offer, the 5th aspect provide is in vannamei boone pair
The positioning of shrimp functional gene, Environment of Litopenaeus vannamei Low population genetic information polymorphism analysis, Environment of Litopenaeus vannamei Low Parentage determination, Environment of Litopenaeus vannamei Low
Application in the structure of genetic map, Environment of Litopenaeus vannamei Low germplasm identification.
Preferably, the application can be used for the prawn functional gene positioning of 2-13 kind Environment of Litopenaeus vannamei Low populations, colony simultaneously
Hereditary information polymorphism analysis, Parentage determination, the structure of genetic map, germplasm identification.
Beneficial effect of the present invention：
(1) technical scheme provided by the invention can identify the heredity of Environment of Litopenaeus vannamei Low LvL1, LvL2, LvL3 microsatellite simultaneously
The variation situation of marked locus, method is easy, and sequencing result can intuitively reflect the gene of each individual of Environment of Litopenaeus vannamei Low
Type.
(2) specific primer provided by the invention for tri- kinds of microsatellite DNA marks of LvL1, LvL2, LvL3, annealing temperature
Degree is 60 DEG C, and the genetic polymorphism of height can be presented in Environment of Litopenaeus vannamei Low population detects.
(3) core sequence (tri- kinds of microsatellite DNAs of LvL1, LvL2, LvL3) of the invention is the expression fragment of functional gene
Sequence, microsatellite locus therein is directly related to functional gene, and its variation situation can reflect this functional gene variation situation.
(4) present invention is mainly applied to Environment of Litopenaeus vannamei Low germ plasm resource and analysis of genetic diversity, Parentage determination, pedigree structure
Build, molecular population genetics, the structure of genetic map, the positioning of important economical trait and the research of functional gene.
Figure of description
Fig. 1 is sequencing of LvL1, LvL2, LvL3 microsatellite DNA mark provided in an embodiment of the present invention in two individuals
Genotyping result；
Fig. 2 is 13 Environment of Litopenaeus vannamei Low population genetics figure provided in an embodiment of the present invention.
Embodiment
As described below is the preferred embodiment of the embodiment of the present invention, it is noted that for the common skill of the art
For art personnel, on the premise of principle of the embodiment of the present invention is not departed from, some improvements and modifications can also be made, these improvement
The protection domain of the embodiment of the present invention is also considered as with retouching.
Unless otherwise noted, agents useful for same and consumptive material are commercial goods in the embodiment of the present invention.
Unless otherwise indicated, the immunology of use of the embodiment of the present invention, biochemistry, chemistry, molecular biology, microorganism
, cell biology, the routine techniques of genomics and recombinant DNA, within the technical ability of this area.Including but not limited to join
See following document：Pehanorm Brooker (Sambrook), not Ritchie (Fritsch) and the Germania base of a fruit this (Maniatis),《Molecule gram
It is grand：Laboratory manual》(MOLECULAR CLONING:A LABORATORY MANUAL), the 2nd editor (1989)；《The present age point
Sub- biological experiment handbook》(CURRENT PROTOCOLS IN MOLECULAR BIOLOGY) (F.M. Austria Su Beier
Et al. (F.M.Ausubel) editor, (1987))；《Enzymology method》(METHODS IN ENZYMOLOGY) series (academic publishing
Company)：《PCR2:Practical approach》(PCR 2:A PRACTICAL APPROACH) (M.J. McPphersons
(M.J.MacPherson), B.D. Hei Musi (B.D.Hames) and Taylor G.R. (G.R.Taylor) edit (1995)), Ha Luo
(Harlow) and in drawing (Lane) edits (1988)《Antibody：Laboratory manual》(ANTIBODIES,A LABORATORY
MANUAL), and《Animal cell culture》(ANIMAL CELL CULTURE) (R.I. Fu Leixieni (R.I.Freshney) are compiled
Collect (1987)).
In the embodiment of the invention, the invention provides a kind of for the more of Environment of Litopenaeus vannamei Low germplasm identification
Weight PCR primer, method and application.The described method for Environment of Litopenaeus vannamei Low germplasm identification, including：All receive is extracted first
Shore prawn genome and dilute it is standby, utilize tri- microsatellite DNA core sequences of Environment of Litopenaeus vannamei Low LvL1, LvL2, LvL3, design
Specific primer simultaneously adds fluorescence labeling FAM (Fluoresceincarboxylic acid), HEX (chlordene fluorescein) and TAMRA (carboxyl tetramethyls respectively
Rhodamine) fluorescence labeling, then using multiple PCR technique, simultaneously to three primer pair Environment of Litopenaeus vannamei Low in a PCR reacts
Genomic DNA enters performing PCR amplification, and amplified production is detected by ABI sequencing analysis instrument to fluorescent label DNA fragment length,
The genotype (being as shown in Figure 1 the genotype of two random individuals) of each individual is determined according to DNA fragmentation size, is used
Popgene softwares carry out the identification and analysis of germ plasm resource (as shown in Fig. 2 and table 2).
In a specific embodiment of the invention, the described method for Environment of Litopenaeus vannamei Low germplasm identification, including but
It is not limited to the one or more steps of following steps：
1. extract Environment of Litopenaeus vannamei Low genome
1) Environment of Litopenaeus vannamei Low muscle 50mg is taken, is placed in 2ml centrifuge tubes, 540 μ l STE lysis buffers are added after shredding
(100mmol/L NaCl, 10mmol/L Tris-Cl, pH 8.0；1mmol/L EDTA, pH 8.0), 60 μ l SDS and 20 μ l
Proteinase K (20mg/ml), mix；
2) 56 DEG C of digestion process, until lysate is clarified, 6000rpm centrifugations 3min；
3) supernatant is taken, adds 500 μ l saturated phenol/chloroform/isoamyl alcohol (25:24:1), gently rock several minutes,
10000rpm centrifuges 10min；
4) supernatant is taken, is repeated once step 3, untill aqueous phase and organic phase are without albumin layer；
5) supernatant is taken, adds 500 μ l chloroform/isoamyl alcohol (24:1) 10min, 10000rpm centrifugations, are gently rocked
10min；
6) take supernatant, add 1/10th volumes NaAc (3mol/L, pH value 5.2) and 4 DEG C of diploid product in advance
Cold absolute ethyl alcohol, about 30min in -20 DEG C of refrigerators is positioned over, 10000rpm centrifugation 10min, nucleic acid is deposited in ttom of pipe；
7) supernatant is abandoned, adds the ethanol of 1ml 70% washing precipitation, 4 DEG C of centrifugation 5min of 8000rpm；
8) abandon alcohol, centrifuge tube is placed on it is drying precipitated in vacuum desiccator, until ethanol all volatilize；
9) 100 μ 1 × TE of l buffer solutions and 5 μ l RNaseA (10mg/ml) are added, 37 DEG C of digestion RNA 30min, -20 DEG C
Save backup.
2. the design of micro-satellite primers
Three EST original sequences of software SSRHunter analyses are searched by microsatellite and obtain core repeat sequence LvL1：
(TCTG) a ... (TCTCTG) b ... (TCTCTG) c ... (TC) d ... (TC) e ... (TCTG) f, abcdef=5-10；LvL2：(CT)
A ... (CT) b, ab=4-10；LvL3：(CT) a, a=7-15.Using primer-design software PeimerPeimer5.0 to above-mentioned EST
Sequence upstream and downstream carries out design of primers, respectively obtains its micro-satellite primers nucleotide sequence：LvL1：Normal chain 5'-
AAGGCATTGGCAATCAGCTGAAGTA-3', minus strand 5'-ACGCACACGTACACTTGTAGACAGA-3'；LvL2：Normal chain 5'-
CGCCCTCTCTCTATTTCTCTCCACCC-3', minus strand 5'-AGAGGGAGCGGAAAGGGGATACAGT-3'；LvL3：Normal chain
5'-TGGGTAGTACGCTATATTTGGGAAGC-3', minus strand 5'-ACAGAGAGAAGGAGGGAGTCAGGCA-3', annealing temperature
Degree：60 DEG C, add fluorescence labeling FAM (Fluoresceincarboxylic acid) HEX (chlordene fluorescein) and TAMRA respectively at the 5' ends of forward primer
(carboxyl tetramethylrhodamine).
The specific primer nucleotide sequence of three kinds of microsatellite DNA sequences is as shown in table 1：
1 three SSR molecular marker information of table
3.PCR is expanded
PCR reaction solutions use TaKaRaTaqTMHot Start Version, reaction solution composition is 0.3 μ l TaKaRa Taq
HS (5U/ μ l), 6 μ l10 × PCR Buffer, 4.8 μ l dNTP Mixture, three kinds of forward and reverse primers in SSR sites respectively add 0.4 μ
M, DNA profiling 100ng, supplied with sterile purified water to 40 μ l.PCR reaction amplification programs are arranged to：94 DEG C of pre-degeneration 4min, 35
Individual circulation：94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, last 72 DEG C of extensions 5min, 16 DEG C preserve.
4. fluorescence is sequenced and data processing
PCR primer is sent in Huada gene company, fluorescent label DNA fragment detected using ABI sequencing analysis instrument,
The DNA fragmentation size separation allele obtained according to detection, Fig. 1 LvL1, LvL2, LvL3 microsatellite DNA mark are at two
Sequencing and typing result in random individual.The gene data for each individual microsatellite locus that parting is obtained is converted into
Popgene software formats, import and population genetic information polymorphism analysis (such as table 2) is carried out in Popgene softwares；Calculate each family
Between genetic distance, contribute (such as Fig. 2) according to each family genetic distance calculated using NTsys softwares；Fig. 2 is 13 all
Receive shore prawn population genetics figure, 13 Environment of Litopenaeus vannamei Low populations can clearly be divided into 5 big monoids, the result and this reality
Apply a specimen in use to carry out source record consistent.Note：The embodiment of the present invention is detected for different Environment of Litopenaeus vannamei Low species, every kind of
Environment of Litopenaeus vannamei Low repeats many experiments and is averaging.As a result show, reflected provided by the present invention for Environment of Litopenaeus vannamei Low germ plasm resource
Fixed multiple PCR primer, method can be used for Environment of Litopenaeus vannamei Low germ plasm resource and analysis of genetic diversity, Parentage determination, pedigree structure
Build, molecular population genetics, the structure of genetic map, the positioning of important economical trait and the research of functional gene.
2. 13 Environment of Litopenaeus vannamei Low population genetic information polymorphism analysis of table
It will be appreciated by persons skilled in the art that the analysis of biological information method that the present invention uses is art technology
The standard biologic information analysis method that personnel use.Know multiple PCR primer group provided by the invention, multiplex PCR system or
Multiplexed PCR amplification program in advance under, those skilled in the art in embodiment genome extraction, primer synthesis, prepare repair
The scope of the present invention should be included using similar or equivalent replacement by adoring the steps such as primer, the high pass sequencing of fluorescence molecule.
Sequence table
<110>Zhongshan University
<120>A kind of multiple PCR primer, method and application for Environment of Litopenaeus vannamei Low germplasm identification
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<141> 2017-09-01
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aaggcattgg caatcagctg aagta 25
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acgcacacgt acacttgtag acaga 25
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<213>Artificial sequence (artificial synthesized)
<400> 3
cgccctctct ctatttctct ccaccc 26
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<211> 25
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<213>Artificial sequence (artificial synthesized)
<400> 4
agagggagcg gaaaggggat acagt 25
<210> 5
<211> 26
<212> DNA
<213>Artificial sequence (artificial synthesized)
<400> 5
tgggtagtac gctatatttg ggaagc 26
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<213>Artificial sequence (artificial synthesized)
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acagagagaa ggagggagtc aggca 25
Claims (10)
- A kind of 1. multiple PCR primer for Environment of Litopenaeus vannamei Low germplasm identification, it is characterised in that the multiple PCR primer Include the primer sets of following three pairs of primers composition：Pair of primers：Sense primer LvL1-F, anti-sense primer LvL1-R；Second pair of primer：Sense primer LvL2-F, anti-sense primer LvL2-R；3rd pair of primer：Sense primer LvL3-F, anti-sense primer LvL3-R；Wherein, sense primer LvL1-F nucleotide sequence include SEQ ID NO.1 shown in nucleotide sequence or with SEQ ID Nucleotide sequence complementary NO.1；Anti-sense primer LvL1-R nucleotide sequence include SEQ ID NO.2 shown in nucleotide sequence or with SEQ ID NO.2 Complementary nucleotide sequence；Sense primer LvL2-F nucleotide sequence include SEQ ID NO.3 shown in nucleotide sequence or with SEQ ID NO.3 Complementary nucleotide sequence；Anti-sense primer LvL2-R nucleotide sequence include SEQ ID NO.4 shown in nucleotide sequence or with SEQ ID NO.4 Complementary nucleotide sequence；Sense primer LvL3-F nucleotide sequence include SEQ ID NO.5 shown in nucleotide sequence or with SEQ ID NO.5 Complementary nucleotide sequence；Anti-sense primer LvL3-R nucleotide sequence include SEQ ID NO.6 shown in nucleotide sequence or with SEQ ID NO.6 Complementary nucleotide sequence.
- 2. a kind of multiplex PCR system for Environment of Litopenaeus vannamei Low germplasm identification, it is characterised in that including claim 1 institute The multiple PCR primer stated.
- 3. it is used for the multiplex PCR system of Environment of Litopenaeus vannamei Low germplasm identification as claimed in claim 2, it is characterised in that institute Terminal modified there is fluorescent tag molecule the 5 of the multiple PCR primer of use.
- 4. it is used for the multiplex PCR system of Environment of Litopenaeus vannamei Low germplasm identification as claimed in claim 2, it is characterised in that draw Thing is modified with FAM to LvL1-F, LvL1-R 5 ends；5 ends of primer pair LvL2-F, LvL2-R are modified with HEX, primer pair LvL3-F, LvL3-R 5 ends are modified with TAM.
- 5. it is used for the multiplex PCR system of Environment of Litopenaeus vannamei Low germplasm identification as claimed in claim 2, it is characterised in that institute State in multiplex PCR system, multiple PCR primer group is when carrying out multiplex PCR, using 60 DEG C of annealing.
- 6. it is used for the multiplex PCR system of Environment of Litopenaeus vannamei Low germplasm identification as claimed in claim 2, it is characterised in that institute State in multiplex PCR system, for multiple PCR primer group when carrying out multiplex PCR, each forward and reverse primer equimolar is than configuration.
- A kind of 7. method for Environment of Litopenaeus vannamei Low germplasm identification, it is characterised in that methods described includes:1) Environment of Litopenaeus vannamei Low genome is obtained；2) Environment of Litopenaeus vannamei Low is obtained using the multiple PCR primer amplification described in claim 1 Genome；3) high-flux sequence is carried out to multiplexed PCR amplification product；4) bioinformatic analysis is carried out to sequencing result, obtained Environment of Litopenaeus vannamei Low germplasm identification result.
- 8. a kind of kit for Environment of Litopenaeus vannamei Low germplasm identification, it is characterised in that including more described in claim 1 Weight PCR primer, in addition to multiplexed PCR amplification reagent.
- 9. a kind of multiple PCR primer, multiplex PCR system, such as claim 7 as claimed in claim 2 as claimed in claim 1 Described method, kit as claimed in claim 8 are positioned in Environment of Litopenaeus vannamei Low functional gene, Environment of Litopenaeus vannamei Low population genetic Information polymorphism analysis, Environment of Litopenaeus vannamei Low Parentage determination, the structure of Environment of Litopenaeus vannamei Low genetic map or Environment of Litopenaeus vannamei Low germplasm money Application in the identification of source.
- 10. application as claimed in claim 9, it is characterised in that multiple PCR primer, such as right as claimed in claim 1 It is required that the multiplex PCR system, method as claimed in claim 7, kit as claimed in claim 8 described in 2 are in vannamei boone The positioning of prawn population functional gene, hereditary information polymorphism analysis, Parentage determination, the structure of genetic map or Environment of Litopenaeus vannamei Low kind Application in the identification of matter resource.
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| CN113151503A (en) * | 2021-05-18 | 2021-07-23 | 中国水产科学研究院黄海水产研究所 | Litopenaeus vannamei microsatellite marked primer combination and application |
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