CN109182546A - For the SSR fluorescent dye primer of pangolin paternity test and application - Google Patents
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- CN109182546A CN109182546A CN201811220793.2A CN201811220793A CN109182546A CN 109182546 A CN109182546 A CN 109182546A CN 201811220793 A CN201811220793 A CN 201811220793A CN 109182546 A CN109182546 A CN 109182546A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses for the SSR fluorescent dye primer of pangolin paternity test and application.The present invention successfully screens and synthesizes 15 pairs of fluorescent marker satellite primers; the primer can be applied to pangolin paternity test; also it can be prepared into the kit for pangolin paternity test; or carry out pangolin Germplasm Identification and family management; to reasonably instruct pangolin artificial breeding to work; for the genetic diversity for protecting pangolin, the researchs such as population genetic mechanism provide strong tool.The present invention also provides a kind of method of pangolin paternity test, addition M13 connector is used in this method, it is only necessary to synthesize a M13 fluorescent dye primer and carry out PCR extension, to save the cost and time for synthesizing that a large amount of fluorescent primers are spent.
Description
Technical field
The present invention relates to Animal Paternity technology object fields, and in particular to the SSR fluorescence for pangolin paternity test
Labeled primer and application.
Background technique
Pangolin is a kind of mammal with atypical morphology feature of mystery, is under the jurisdiction of Mammalia (Mammalia)
Pholidota (Pholidota) pangolin section's (Manidae) pangolin category (Manis).For a long time, people are to pangolin scales
Medicinal demand causes the illegal trading of pangolin to get worse, in addition its habitat is destroyed on a large scale, so that the open country of pangolin
Raw population quantity sharply declines, at present existing 8 types (Gaubert and Antunes, 2005；Zhang et al.,
2015) it is put into endangered wildlife international trade protection catalogue (Convention on international trade
in endangered species of wild fauna and flora I,CITES I)。
Currently, to make the pangolin in Small Population that close relative easily occur numerous for the fragmentation and population isolation of habitat
The problems such as growing, the degeneration of its germplasm, growth performance and fertility is caused to decline is very prominent.And the breeding potential of pangolin compared with
It is low, it is difficult to restore after individual amount is die-offed in Small Population, these Small Populations being isolated from each other easily become extinct in some areas.Although
In-situ protection is the important component of endangered species protection work, but in order to expand the scale of recent species group, increase heredity
Diversity, artificial breeding and in situ conservation have become the important measures of protection endangered species.Item of the pangolin in artificial feeding
Under part, it is ensured that obtain comprehensive, sufficient food and nutrition, obtain good health care, natural enemy is avoided to injure.Cause
This, establishes a stable artificial feeding population, avoids close breeding decline and genetic drift, increases effective population size, with
It takes and carries out the breeding for the purpose of long-term gene exchanges with the smallest individual of affiliation and optimize pairing work to seem especially heavy
It wants.It not only can satisfy the social demand for carrying out scientific research and public education both at home and abroad in this way, but also can be captive breeding individual
Release creates certain condition to supplement wild stocks.
The paternity test of early stage regard blood group or enzyme type etc. as genetic marker.Currently, DNA molecular marker is instead of biography
The genetic marker of system becomes important genetic marker used in paternity test research.Microsatellite marker due to its rich polymorphism,
Relatively conservative single-copy sequence is presented in sequence both ends, and allele number alterable height, heterozygosity is high, genetic stability compared with
It is good, it is in codominant inheritance, therefore be widely used in building genome, the determination of genetic affinity, individual or group's relationship
Among relationship identification.
Microsatellite DNA (Microsatellite) be also known as simple repeated sequence (Simple Sequences Repeats,
SSR), refer to a kind of recurring unit in the repetitive sequence of 2-6 base.There is SSR molecular marker polymorphism height, codominance to lose
It passes, select the advantages that neutral, easily operated, acquisition result is reliable, method is simple, time saving and energy saving, develops into a kind of pole quickly
Have the genetic marker of value, and be widely used in parenthood determination between individual, population genetic analysis, genetic disease are examined
The research fields such as the disconnected, assignment of genes gene mapping, genetic map.
Currently, microsatellite molecular marker to be applied to the report of pangolin paternity test and genetic management, this hair not yet
It is bright to be intended to establish Manis javanica paternity test technology using microsatellite fluorescent marker, to solve in pangolin breeding process, because
The problems such as individual identification is difficult caused by Small Population mating, different parent's mixed breedings, and pedigree is unclear, are established for pangolin farm
Its family pedigree provides scientific basis, to reasonably instruct the breeding child care of pangolin Artificial Population to work.
Summary of the invention
The purpose of the present invention is to provide the SSR fluorescent dye primer for pangolin paternity test, which can be used for
Pangolin paternity test.
Another object of the present invention is that providing answering for the SSR fluorescent dye primer for pangolin paternity test
With carrying out paternity test including the use of the primer pair pangolin, or be prepared into for pangolin paternity test using the primer
Kit.
The present invention is achieved through the following technical solutions:
Mixing sample is knitted according to pangolin multiple groups and obtains its transcript profile, according to the microsatellite locus provided in transcript profile data
Design primer；Then using pangolin saliva sample DNA as template, PCR amplification, preliminary screening are carried out using microsatellite marker primer
The polymorphism of PCR product obtains the high microsatellite marker primer of polymorphism；The high microsatellite marker primer of polymorphism is carried out
FAM fluorescent decoration carries out PCR analysis using pangolin saliva sample DNA as template, carries out genotype judgement to PCR product；Root
Exclusive method is used in combination according to genotyping result and likelihood method carries out the identification of parent child relationship.
Specifically, being used for the SSR fluorescent dye primer of pangolin paternity test, comprising:
M13MJ-F2F:TTTCATACCGGGAAGTCCAC；R:ATGGTCCTAACACCACGGAG；
M13MJ-F3F:CACCTGCATGTACCCCTTTT；R:CCCCCTCAAAATACCACCTT；
M13MJ-F4F:GAGAGAAAGGGGAAAATCGG；R:TGATAGGATGTGAGGAGGGG；
M13MJ-F5F:TGGGGTCTGCTGTTTTCATT；R:CTCCCTCTGTAGGTTGCCCT；
M13MJ-F7F:AGAAGTGATTTGCACCCCTG；R:CAGTGGCCAGAATGGAGATT；
M13MJ-F8F:GTCATGCATGGAAGACAGGA；R:GTCTGGCTGAAATGGAAAGG；
M13MJ-F9F:CCCTATGAGGTGGGCACTAA；R:AACTCCATCAAAGGTGTGGC；
M13MJ-F10F:CCCAGATCCAAAATGAATGG；R:TGCTGATGTTCACTCTTGCC；
M13MJ-F11F:ATCCACCTAGGAACCTCAGC；R:GACTCTTCGGGATTTCACACA；
M13MJ-F12F:ATCCACCTAGGAACCTCAGC；R:GACTCTTCGGGATTTCACACA；
M13MJ-F16F:TTCCCTTCATCTGTTTTGCC；R:CAGCACTGCCAAGGTCTGTA；
M13MJ-F17F:GTAATGGGGTATGTGGTGGG；R:TCCCTGTTCAAACGGAATTT；
M13MJ-F20F:CAGTGCTCATCACATAGCAGG；R:CATGCCTAGTGTTTCACGTTG
M13MJ-F24F:TTCAGCCAGGGTCTCTCAGT；R:TGGGGTTTTTCCTCAATCTG；
M13MJ-F25F:CCAGAGAAAGGTAGGAGCCA；R:TCCAGAAAACAGACCCAAGG.
Further, the forward primer of connection M13 connector, 5 ' end of synthesis plus M13 connector is held to the above primer pair forward direction 5 ',
M13 joint sequence is CACGACGTTGTAAAACGAC；To the end of forward direction 5 ' the connection fluorophor of universal primer M13, fluorescence is synthesized
The M13 forward primer of base group modification, the forward primer sequence of the universal primer M13 are CACGACGTTGTAAAACGAC.Institute
The fluorophor stated is FAM.
The application of SSR fluorescent dye primer for pangolin paternity test of the invention is carried out including the use of the primer
Pangolin paternity test, or it is prepared into the kit for pangolin paternity test using the primer, or carry out using the primer
Germplasm Identification and family management.
A kind of method of pangolin paternity test, comprising the following steps:
A, synthesize above-mentioned 15 primer pairs (M13MJ-F2, M13MJ-F3, M13MJ-F4, M13MJ-F5, M13MJ-F7,
M13MJ-F8、M13MJ-F9、M13MJ-F10、M13MJ-F11、M13MJ-F12、M13MJ-F16、M13MJ-F17、M13MJ-
F20, M13MJ-F24, M13MJ-F25) forward direction 5 ' is held plus the forward primer of M13 connector, the M13 joint sequence are
CACGACGTTGTAAAACGAC；Synthesize the forward primer of the end of forward direction 5 ' the addition fluorophor of universal primer M13；Described is logical
It is CACGACGTTGTAAAACGAC with the forward primer sequence of primer M13.
B, pangolin DNA is extracted as template, and the forward direction using above-mentioned 15 primer pair forward directions 5 ' end plus M13 connector is drawn
Object, reverse primer, universal primer M13 forward direction 5 ' end addition fluorophor forward primer, carry out PCR amplification, to PCR product
Genotype judgement is carried out, number of alleles, observation heterozygosity and desired heterozygosity are calculated using Cervus software, breathe out enlightening Weinberg
Balance check, polymorphism information content, independent elimination factor and accumulation elimination factor, is used in combination exclusive method according to genotyping result
The identification of parent child relationship is carried out with likelihood method.
In above-mentioned pangolin paternity test method, PCR amplification program uses touchdown PCR, actual conditions are as follows: 95 DEG C of pre- changes
Property 10min；95 DEG C of denaturation 15s；From 62 to 52 DEG C of annealing temperature 30s, every 2 DEG C of 2 cycle down annealing temperatures, 72 DEG C of extensions
2min；95 DEG C of denaturation 15s；52 DEG C of annealing temperature 30s；72 DEG C of extension 2min；30 circulations；72 DEG C of extension 10min.
Compared with prior art, novelty of the invention and beneficial effect are:
(1) SSR fluorescent dye primer provided by the invention can be used for Manis javanica paternity test and family management, from
And pangolin artificial breeding is reasonably instructed to work, for the genetic diversity for protecting pangolin, the researchs such as population genetic mechanism are mentioned
Strong tool is supplied；
(2) present invention is using the method for adding M13 connector, it is only necessary to synthesize a M13 fluorescent dye primer and carry out PCR expansion
Exhibition, to save the cost and time for synthesizing that a large amount of fluorescent primers are spent.
Detailed description of the invention
Fig. 1 is the Fluorescence PCR schematic diagram for adding M13 connector.
Specific embodiment
The following examples are further illustrations of the invention, rather than limiting the invention.
The screening of 1 Manis javanica microsatellite marker of embodiment
Solution takes Manis javanica (dead) tongue, stomach, large intestine, pancreas, liver, saliva equal samples and carries out mixed grinding,
Total serum IgE is extracted to be sequenced, splice, assemble and search microsatellite locus for transcript profile.18693 sites SSR are detected altogether, wherein
Mononucleotide repeat sequence 12120, dinucleotides repetitive sequence 3202, trinucleotide repeats sequence 1594, four nucleosides
Sour repetitive sequence 259, fermentation by five tubes 52, Hexanucleotide repetitive sequence 23.According to SSR obtained
Point designs 56079 pairs of primers using Primer 5.
35 are extracted using animal tissue's DNA Mini Kit (Hipure Tissue DNA Mini Kit, Magen)
Only (female 15, male 20) Manis javanica saliva sample DNA, NanoDropDN-1000 UV detector detection
DNA concentration and quality；Obtained DNA is diluted to the working solution of 100ng/ μ l with distilled water, -20 DEG C save backup.
Using Manis javanica saliva DNA as template, in acquired micro-satellite primers, randomly choose 40 pairs of primers and
Its forward direction 5 ' end addition M13 sequence (CACGACGTTGTAAAACGAC) is synthesized, and PCR amplification system is 10 μ l, including
The 1 μ l of forward primer of 1 μ l, 2 × PCR Mix of 100ng/ μ l DNA profiling, 52 μ l of μ l, ddH2O, 5 ' ends plus M13 connector, reversely
1 μ l of primer.Amplified reaction program uses touchdown PCR (touchdown PCR): 95 DEG C of initial denaturation 10min；95 DEG C of denaturation 15s；It moves back
Fiery from 62 to 52 DEG C of temperature 30s, every 2 DEG C of 2 cycle down annealing temperatures, 72 DEG C of extension 2min；95 DEG C of denaturation 15s；52 DEG C of annealing
Temperature 30s；72 DEG C of extension 2min；30 circulations；72 DEG C of extension 10min, last 4 DEG C of preservations.Utilize polyacrylamide gel electricity
Swimming detection PCR product, preliminary analysis filter out 15 pairs of primers (table 1) with polymorphic site.
The Manis javanica micro-satellite primers sequence information with polymorphism is screened in 1 present invention of table
2 polymorphic micro-satellite site PCR amplification of embodiment and Genotyping
(1) microsatellite fluorescent dye primer PCR reacts
Universal primer M13 forward direction 5 ' end addition FAM fluorophor modified (Boutin-Ganache et al.,
2001) the M13 forward primer of FAM modification, is obtained, M13 forward primer sequence is CACGACGTTGTAAAAC GAC；To embodiment
1 preliminary analysis filters out the end of forward direction 5 ' the addition M13 sequence of 15 pairs of primers with polymorphic site
(CACGACGTTGTAAAACGAC) forward primer of 5 ' end of synthesis plus M13 connector.
The 35 Manis javanica saliva sample DNA obtained, which are extracted, as template using embodiment 1 carries out Fluorescence PCR,
Amplification system is 10 μ l, including 1 μ l, 2 × PCR Mix of 100ng/ μ l DNA profiling, 5 1.6 μ l of μ l, ddH2O, has polymorphism
The M13 of the end of forward direction 5 ' of 15 pairs of primers in site plus the 0.4 μ l of forward primer of M13 connector, 1 μ l, FAM modification of reverse primer are positive
(reaction principle is shown in Fig. 1 to 1 μ l of primer, bibliography: Boutin-Ganache et al., 2001.M13-Tailed Primers
Improve the Readability and Usability of Microsatellite Analyses Performed
with Two Different Allele-Sizing Methods,Biotechniques,31(1):25-28).Amplified reaction
Program uses touchdown PCR (touchdown PCR): 95 DEG C of initial denaturation 10min；95 DEG C of denaturation 15s；Annealing temperature 30s from 62 to
52 DEG C, every 2 DEG C of 2 cycle down annealing temperatures, 72 DEG C of extension 2min；95 DEG C of denaturation 15s；52 DEG C of annealing temperature 30s；72 DEG C of extensions
2min；30 circulations；72 DEG C of extension 10min, last 4 DEG C are kept in dark place.
The PCR product that fluorescent primer has expanded identifies its concentration with 1% agarose gel electrophoresis, is diluted to suitable capillary
The concentration of electrophoresis tube detection, is added GS500LIZ internal standard used in capillary electrophoresis analysis, ABI3730XL high pass is put into after processing
It measures DNA sequencer and carries out Capillary Electrophoresis and STR analysis to determine equipotential base of the pangolin microsatellite marker in Different Individual
Because of size.
(2) analysis of genetic diversity
Number of alleles (k) is calculated using Cervus software, observation heterozygosity (Ho) and desired heterozygosity (He), Ha Diwen
Burger balances (HWE) and examines, and the elimination factor of polymorphism information content (PIC) and candidate parent describes Manis javanica phase with this
Close the feature of Genetic Polymorphism of Microsatellite DNA.
15 microsatellite genetic marker diversity analysis the results are shown in Table 2,15 and micro- defend in 35 Manis javanica DNA samples
The nucleotide sequence of championship point is respectively as shown in SEQ ID NO.1-SEQ ID NO.15.Wherein, primer M13MJ-F2 is amplified
Microsatellite locus nucleotide sequence as shown in SEQ ID NO.1；The core for the microsatellite locus that primer M13MJ-F3 is amplified
Nucleotide sequence is as shown in SEQ ID NO.2；Wherein, the nucleotide sequence for the microsatellite locus that primer M13MJ-F4 is amplified is such as
Shown in SEQ ID NO.3；Wherein, the nucleotide sequence for the microsatellite locus that primer M13MJ-F5 is amplified such as SEQ ID NO.4
It is shown；Wherein, the nucleotide sequence for the microsatellite locus that primer M13MJ-F7 is amplified is as shown in SEQ ID NO.5；Wherein, draw
The nucleotide sequence for the microsatellite locus that object M13MJ-F8 is amplified is as shown in SEQ ID NO.6；Wherein, primer M13MJ-F9 expands
Increase the nucleotide sequence of microsatellite locus out as shown in SEQ ID NO.7；Wherein, what primer M13MJ-F10 was amplified micro- defends
The nucleotide sequence of championship point is as shown in SEQ ID NO.8；Wherein, the core for the microsatellite locus that primer M13MJ-F11 is amplified
Nucleotide sequence is as shown in SEQ ID NO.9；Wherein, the nucleotide sequence for the microsatellite locus that primer M13MJ-F12 is amplified is such as
Shown in SEQ ID NO.10；Wherein, the nucleotide sequence for the microsatellite locus that primer M13MJ-F16 is amplified such as SEQ ID
Shown in NO.11；Wherein, the nucleotide sequence for the microsatellite locus that primer M13MJ-F17 is amplified is as shown in SEQ ID NO.12；
Wherein, the nucleotide sequence for the microsatellite locus that primer M13MJ-F20 is amplified is as shown in SEQ ID NO.13；Wherein, primer
The nucleotide sequence for the microsatellite locus that M13MJ-F24 is amplified is as shown in SEQ ID NO.14；Wherein, primer M13MJ-F25
The nucleotide sequence of the microsatellite locus amplified is as shown in SEQ ID NO.15.
As shown in Table 2,15 microsatellite locus detect that number of alleles range is 4-11, average allele number
It is 8；Heterozygosity is observed between 0.206-0.857, it is expected that heterozygosity is between 0.454-0.869；Polymorphism information content
(PIC) value is between 0.413-0.84.Primer heredity with higher to prove the microsatellite locus of the invention screened is more
State property.When Parent hereditary information is unknown, excludes filial generation and assume that parent is the independent elimination factor (NE-PP) of parent child relationship
For 0.117-0.599；When known to one side's hereditary information of parent, excludes filial generation and assume that parent is the independent exclusion of parent child relationship
Rate (NE-2P) is 0.317-0.752；When Parent both sides hereditary information is known, excludes filial generation nothing to do with and assume parent couple
The independent elimination factor (NE-1P) for being parent child relationship is 0.53-0.896.Accumulation probability of exclusion NE-1P, NE-2P, NE-PP are respectively
0.9999、0.9999、0.995。
The polymorphisms characteristic of 2 15 microsatellite locus of Manis javanica of table
| Locus | k | N | HObs | HExp | PIC | NE-1P | NE-2P | NE-PP | HW |
| M13MJ-F2 | 6 | 35 | 0.657 | 0.706 | 0.644 | 0.721 | 0.553 | 0.373 | NS |
| M13MJ-F3 | 6 | 33 | 0.606 | 0.74 | 0.682 | 0.686 | 0.513 | 0.333 | ND |
| M13MJ-F4 | 5 | 34 | 0.382 | 0.65 | 0.6 | 0.764 | 0.588 | 0.397 | NS |
| M13MJ-F5 | 8 | 32 | 0.469 | 0.692 | 0.645 | 0.714 | 0.534 | 0.334 | NS |
| M13MJ-F7 | 11 | 35 | 0.743 | 0.853 | 0.821 | 0.485 | 0.317 | 0.145 | ND |
| M13MJ-F8 | 11 | 32 | 0.469 | 0.869 | 0.84 | 0.446 | 0.285 | 0.117 | ND |
| M13MJ-F9 | 4 | 34 | 0.206 | 0.454 | 0.413 | 0.896 | 0.752 | 0.599 | ND |
| M13MJ-F10 | 9 | 35 | 0.857 | 0.814 | 0.777 | 0.556 | 0.38 | 0.195 | ND |
| M13MJ-F11 | 9 | 35 | 0.6 | 0.822 | 0.785 | 0.546 | 0.37 | 0.189 | ND |
| M13MJ-F12 | 7 | 35 | 0.429 | 0.78 | 0.737 | 0.616 | 0.435 | 0.246 | ND |
| M13MJ-F16 | 7 | 34 | 0.382 | 0.673 | 0.611 | 0.751 | 0.584 | 0.403 | NS |
| M13MJ-F17 | 11 | 35 | 0.657 | 0.82 | 0.789 | 0.53 | 0.355 | 0.166 | ND |
| M13MJ-F20 | 9 | 32 | 0.625 | 0.825 | 0.789 | 0.537 | 0.362 | 0.179 | ND |
| M13MJ-F24 | 6 | 32 | 0.656 | 0.709 | 0.654 | 0.713 | 0.538 | 0.352 | NS |
| M13MJ-F25 | 8 | 34 | 0.735 | 0.743 | 0.692 | 0.669 | 0.491 | 0.302 | NS |
Note: N is detection individual of sample number；K is number of alleles；Hobs is observation heterozygosity；HExp is desired heterozygosity；
HW is hardy weinberg equilibrium inspection；PIC is polymorphism information content.
It is generally believed that the number of alleles of microsatellite marker could preferably be used for the heredity point of species when being at least 4
Analysis and paternity test.The present invention detects 117, allele altogether, and average allele is 8, and average observation heterozygosity is
0.565, average expectation heterozygosity is 0.743, and the lesser difference of the two illustrates that this 15 microsatellite markers have preferable gene
Heterozygosity accurate can reflect the hereditary feature of group.When only accumulation elimination factor is greater than 0.8, microsatellite mark is utilized
The parent child relationship of note identification just has certain reliability, in the present invention, 15 microsatellite markers accumulate elimination factors it is equal > 0.99.
It micro- is defended in conclusion the present invention has developed Manis javanica 15 microsatellite locus with polymorphism and expands this 15
The primer sequence and amplification method of championship point can be applied to the genetic diversity Journal of Sex Research of Manis javanica different geographic populations, weight
Renaturation is good, is a kind of reliable and effective molecular labeling.
The application microsatellite marker of embodiment 3 carries out the applicating example of Manis javanica paternity test
Exclusive method and likelihood method is used in combination using above-mentioned 15 microsatellite markers, parent-offspring is carried out to 35 Manis javanicas
Relationship identification, and the result identified in person is compared with feed lot pedigree record, to verify the accuracy of this method.It utilizes
It is 5.75 that 3.0 software of CERVUS, which calculates LOD value in 95% confidence level, is 2 in 80% confidence level.When confidence level is 95%,
Number 246 and C46 are identified, A26 and BB are mother-child relationship (MCR)；Identifying A25 and BB is set membership.Will in person qualification result with
Pedigree Record Comparison, obtained accuracy rate are 100%.
The above is only the preferred embodiment of the present invention, it is noted that above-mentioned preferred embodiment is not construed as pair
Limitation of the invention, protection scope of the present invention should be defined by the scope defined by the claims..For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, several improvements and modifications can also be made, these change
It also should be regarded as protection scope of the present invention into retouching.
Sequence table
<110>Guangdong Province's living resources Applied Research Laboratory
<120>for the SSR fluorescent dye primer of pangolin paternity test and application
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 200
<212> DNA
<213>artificial sequence (M13MJF2)
<400> 1
ttttcatacc gggaagtcca ctgactacag gagactacta gttaggtttc taggtttctg 60
cgatcttgac ccaccttcca tccatccatc catccatcca tccatccatc cagccagcca 120
gccagccatt caattaaaaa tgtattggaa ctccgtggtg ttaggaccat gatcaggcat 180
tcttcctctg aaagatgata 200
<210> 2
<211> 230
<212> DNA
<213>artificial sequence (M13MJF3)
<400> 2
ccccttttct ctcttatgtg tcgtacctgg tttagatgac aattcatatg atcactctag 60
tcaagagtag ctggttgatt tttatttatt tatttattta tttatttatt tattttaaac 120
actgctggag aaaactgtga tgatgattgt atcttaagtg aatgatggcc ataatgggaa 180
aaacagaaaa agagaaggtg gtattttgag ggggaaaggt gggcataaat 230
<210> 3
<211> 249
<212> DNA
<213>artificial sequence (M13MJF4)
<400> 3
tgtgaaatag gagagaaagg ggaaaatcgg taaagaattc attcattcat tcattcattc 60
attcattcaa catatttatt gagacctcat aaatagcaga aataatccca aatacttagc 120
atatggcagt gaacaagaga gccaaaaatc cctctcctag gtaagcctgt gtgtgacagc 180
gggtgtaggc aggaacaatt acaaagacac atgaaacata tctgctgccc ctcctcacat 240
cctatcagc 249
<210> 4
<211> 219
<212> DNA
<213>artificial sequence (M13MJF5)
<400> 4
tggggtctgc tgttttcatt atagaatact tttagttaag acaaggaaaa gtgaaaggaa 60
cagaagtctc tttttaatag tccatatgaa aatctcatat gcaattaatt aattaattaa 120
ttaattaatt aattaatatt tctctaaggt ctcctggctt ctgaaggttt atttagggca 180
acctacagag ggaggaagaa ataactgtga ctcatgttc 219
<210> 5
<211> 239
<212> DNA
<213>artificial sequence (M13MJF7)
<400> 5
agtgatttgc acccctgagg aaaggcctga cttctttttc ggtcctctcc tccttgttct 60
tatttctgtc tgtttgtttg ttcattttct cattcattca ttcattcatt cattcattca 120
ttcattcatt cattcattca ttcccatttg ctgcctcaag tgcacccact ttctcatggc 180
tgcacagggt ttaagaggtg actttaatct ccattctggc cactgggtcc atcatccca 239
<210> 6
<211> 250
<212> DNA
<213>artificial sequence (M13MJF8)
<400> 6
gtcatgcatg gaagacagga ctaaggcaaa tgctattatg attagagcac tccaggtcat 60
gacactcact ggatattgtt ttttgtttgt ttgtttgttt gtttgtttgt ttgttttgtt 120
tcaaagctat tacaactaag ggatgatgtg ctcctctttt tccttaccag taaacatttt 180
agtggtagtg gtatattaaa tagcaaaaag ttagtggcca tccctttcca tttcagccag 240
actaacccat 250
<210> 7
<211> 300
<212> DNA
<213>artificial sequence (M13MJF9)
<400> 7
ccctatgagg tgggcactaa tgttatttcc atgtcacaga tgaggaaaca gaggaccagc 60
aaggtacagt cagttgccca ggccacacag ccaggatttc agccctaaag tctgattcca 120
gagtttgagt tctccattga gccctaccgt cgtttggatg aatgaatgaa tgaatgaatg 180
aatgaatgag tgaatgaagc ccccggggag gggacagcag ggcccccact cctgcttaca 240
gcctttccca ccagctgcca cacctttgat ggagttttcc cagaggggcc tgtcttggga 300
<210> 8
<211> 210
<212> DNA
<213>artificial sequence (M13MJF10)
<400> 8
cccagatcca aaatgaatgg agccggaaat tacctcatca gttggtatta atcagccaag 60
caattgacag gctgattgac aggctgggag cccctgaggc tgttgacaaa ggaacatgtt 120
gcttctggaa tgaatgaatg aatgaatgaa tgaatgaatg gggcttgatg tttgatactg 180
tggaagtaag gcaagagtga acatcagcaa 210
<210> 9
<211> 260
<212> DNA
<213>artificial sequence (M13MJF11)
<400> 9
atccacctag gaacctcagc taacagcaac tttctaggtc tctgagttga ttgatttaac 60
atgttaaata gatatagata gatagatgga tagataatag atagatgata gatgatagat 120
acatagatga tagatgatag atgatagata gatagataga tagatagata gatagataga 180
tagatagata gaccaactga tcttgaaatg tgtgcatagt ttgtaaactg tgtgaaatcc 240
cgaagagtct ttgaagagaa 260
<210> 10
<211> 249
<212> DNA
<213>artificial sequence (M13MJF12)
<400> 10
atccacctag gaacctcagc taacagcaac tttctaggtc tctgagttga ttgatttaac 60
atgttaaata gatatagata gatagatgga tagataatag atagatgata gatgatagat 120
acatagatga tagatgatag atagatagat agatagatag atagatagat agatagatag 180
atagaccaac tgatcttgaa atgtgtgcat agtttgtaaa ctgtgtgaaa tcccgaagag 240
tctttgaag 249
<210> 11
<211> 249
<212> DNA
<213>artificial sequence (M13MJF16)
<400> 11
ttcccttcat ctgttttgcc cattctccta acccacccag tttgttctct acatttatga 60
gcatttttct ggttggttgg ttggttggtt ggttggttgg ttggttggtt gttggttgtc 120
ttgtttaaaa aaaagaaggg taaaaccata tctttggaaa tcaaataaca ctgaaaaaaa 180
tgctgatctg aagatgatgc acttacacca tctagaaaat tgtacagacc ttggcagtgc 240
tgccctatc 249
<210> 12
<211> 219
<212> DNA
<213>artificial sequence (M13MJF17)
<400> 12
ctgtaatggg gtatgtggtg gggacttgat aatgggggga gtcagtaacc ataatgttgc 60
tcatgtgatt gtatattaat ggtaccataa taaataaata gatagataga tagatagata 120
gatagataga tagatagata gatagataat ctgcagcgtt atccatataa gtagtttgct 180
gagagagtat aattgaaatt ccgtttgaac agggaagat 219
<210> 13
<211> 199
<212> DNA
<213>artificial sequence (M13MJF20)
<400> 13
cagtgctcat cacatagcag gtactcagaa agtaaaatta gttcccttct tttagcctta 60
cttgatcata accattggag atagatagat agatagatag atagatagat agatagatag 120
atagatagat agatagatag atagcagtaa gagatacaac gtgaaacact aggcatgtaa 180
attctcaaag aaaacaatt 199
<210> 14
<211> 199
<212> DNA
<213>artificial sequence (M13MJF24)
<400> 14
ttcagccagg gtctctcagt gactgctgag tagaacactc tgaaggtcca tgtcagtccc 60
atgaaataag aatgagaaag agactttgta tattaacctt ctgatacttt ggtgctcttt 120
tttgttttgt tttgttttgt tttgttttgt tttgttatag caacataaca gattgaggaa 180
aaaccccaaa tactcatgc 199
<210> 15
<211> 141
<212> DNA
<213>artificial sequence (M13MJF25)
<400> 15
ccagagaaag gtaggagcca aaagctgggg gaaacaaaac aaaacaaaac aaaacaaaac 60
aaaacaaaac aaaacaacac tttgcagcct ctaattctgg gagtaaaacc ttgggtctgt 120
tttctggagt gaggagggaa g 141
Claims (5)
1. being used for the SSR fluorescent dye primer of pangolin paternity test, comprising:
M13MJ-F2 F:TTTCATACCGGGAAGTCCAC；R:ATGGTCCTAACACCACGGAG；
M13MJ-F3 F:CACCTGCATGTACCCCTTTT；R:CCCCCTCAAAATACCACCTT；
M13MJ-F4 F:GAGAGAAAGGGGAAAATCGG；R:TGATAGGATGTGAGGAGGGG；
M13MJ-F5 F:TGGGGTCTGCTGTTTTCATT；R:CTCCCTCTGTAGGTTGCCCT；
M13MJ-F7 F:AGAAGTGATTTGCACCCCTG；R:CAGTGGCCAGAATGGAGATT；
M13MJ-F8 F:GTCATGCATGGAAGACAGGA；R:GTCTGGCTGAAATGGAAAGG；
M13MJ-F9 F:CCCTATGAGGTGGGCACTAA；R:AACTCCATCAAAGGTGTGGC；
M13MJ-F10 F:CCCAGATCCAAAATGAATGG；R:TGCTGATGTTCACTCTTGCC；
M13MJ-F11 F:ATCCACCTAGGAACCTCAGC；R:GACTCTTCGGGATTTCACACA；
M13MJ-F12 F:ATCCACCTAGGAACCTCAGC；R:GACTCTTCGGGATTTCACACA；
M13MJ-F16 F:TTCCCTTCATCTGTTTTGCC；R:CAGCACTGCCAAGGTCTGTA；
M13MJ-F17 F:GTAATGGGGTATGTGGTGGG；R:TCCCTGTTCAAACGGAATTT；
M13MJ-F20 F:CAGTGCTCATCACATAGCAGG；R:CATGCCTAGTGTTTCACGTTG
M13MJ-F24 F:TTCAGCCAGGGTCTCTCAGT；R:TGGGGTTTTTCCTCAATCTG；
M13MJ-F25 F:CCAGAGAAAGGTAGGAGCCA；R:TCCAGAAAACAGACCCAAGG.
2. application of the primer described in claim 1 in pangolin paternity test.
3. primer described in claim 1 is preparing the application in pangolin paternity test kit.
4. a kind of method of pangolin paternity test, which comprises the following steps:
A, the forward primer of synthesis claim 1 primer pair forward direction 5 ' end plus M13 connector, the M13 joint sequence are
CACGACGTTGTAAAACGAC；Synthesize the forward primer of the end of forward direction 5 ' the addition fluorophor of universal primer M13；Described is logical
It is CACGACGTTGTAAAACGAC with the forward primer sequence of primer M13；
B, pangolin DNA is extracted as template, using the forward primer of the end of claim 1 primer pair forward direction 5 ' plus M13 connector, instead
The forward primer of addition fluorophor is held to the forward direction 5 ' of primer, universal primer M13, carries out PCR amplification, and PCR product is carried out
Genotype determines, calculates number of alleles, observation heterozygosity and desired heterozygosity, hardy weinberg equilibrium using Cervus software
Examine, polymorphism information content, independent elimination factor and accumulation elimination factor, according to genotyping result be used in combination exclusive method with seemingly
Right method carries out the identification of parent child relationship.
5. the method for pangolin paternity test according to claim 4, which is characterized in that the PCR amplification program is adopted
With touchdown PCR, actual conditions are as follows: 95 DEG C of initial denaturation 10min；95 DEG C of denaturation 15s；From 62 to 52 DEG C of annealing temperature 30s, every 2
2 DEG C of cycle down annealing temperature, 72 DEG C of extension 2min；95 DEG C of denaturation 15s；52 DEG C of annealing temperature 30s；72 DEG C of extension 2min；30
Circulation；72 DEG C of extension 10min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111172241A (en) * | 2019-12-10 | 2020-05-19 | 吉林省农业科学院 | Sheep four-base repeated microsatellite marker and screening method, primer set and application thereof |
| CN111662989A (en) * | 2020-06-16 | 2020-09-15 | 广东省生物资源应用研究所 | SSR fluorescence labeling primer for paternity test of deer and identification method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111172241A (en) * | 2019-12-10 | 2020-05-19 | 吉林省农业科学院 | Sheep four-base repeated microsatellite marker and screening method, primer set and application thereof |
| CN111172241B (en) * | 2019-12-10 | 2022-11-22 | 吉林省农业科学院 | Sheep four-base repeated microsatellite marker and screening method, primer set and application thereof |
| CN111662989A (en) * | 2020-06-16 | 2020-09-15 | 广东省生物资源应用研究所 | SSR fluorescence labeling primer for paternity test of deer and identification method |
| CN111662989B (en) * | 2020-06-16 | 2022-05-10 | 广东省科学院动物研究所 | SSR fluorescence labeling primer for paternity test of deer on slope and identification method |
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