CN108753987A - A kind of method of the general microsatellite Multiplex fluorescent PCR of silver carp flathead - Google Patents
A kind of method of the general microsatellite Multiplex fluorescent PCR of silver carp flathead Download PDFInfo
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- CN108753987A CN108753987A CN201810557528.7A CN201810557528A CN108753987A CN 108753987 A CN108753987 A CN 108753987A CN 201810557528 A CN201810557528 A CN 201810557528A CN 108753987 A CN108753987 A CN 108753987A
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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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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The present invention relates to a kind of method of the general microsatellite Multiplex fluorescent PCR of silver carp flathead, belong to aquatic livestock Germplasm Identification technical field in aquaculture field.It is collected first collects silver carp flathead fin ray sample, and genomic DNA is extracted using the method for traditional phenol-chloroform, primer pair is then related to according to the requirement of purpose expanding fragment length difference, choose amplified band clearly 3 group of 4 heavy PCR system, it is related to 12 microsatellite markers altogether, fluorescent decoration finally is carried out to primer and amplification is verified.Multiple PCR method of the present invention, have the characteristics that it is swift to operate, be easy to implement, be low-cost, a kind of new molecular labeling ancillary technique can be provided for researchs such as silver carp flathead blastogenesis assessment and family selective breedings.
Description
Technical field
The present invention relates to a kind of methods of the general microsatellite Multiplex fluorescent PCR of silver carp flathead, are suitable for silver carp flathead Population variation
With the researchs such as paternity test, belong to aquatic livestock Germplasm Identification technical field in aquaculture field.
Background technology
Silver carp (Hypophthalmichthys molitrix) and flathead (Hypophthalmichthys nobilis) are Cyprinidae
Nearly edge species in fish are distributed widely in as the important indigenous strains of China in major rivers and lakes.Both belong to and considers feeding habits
Fish are the important living resources of water purification fishery, have the important ecological value in current water environment protection and in administering.Meanwhile
The two has long cultivation history, the cultivation scale of construction is big, in CHINESE FRESHWATER fishing as the large freshwater fish culturing kind of Chinese tradition
Occupy extremely important economic status in industry.Therefore, the effective protection to silver carp flathead germ plasm resource and utilization are of great significance.So
And influenced by building of irrigation works, environmental pollution, overfishing etc., the wild resource of silver carp and flathead is subject to high impact, in recent years
Wild resource amount is in drastically downward trend.Further, since the features such as adult fish individual is big, the breeding cycle is long, the selection and breeding of two kinds of fishes are real
It is larger to apply difficulty, breeding coverage rate is relatively low in its aquaculture, and parental source is larger to the degree of dependence of wild resource.Currently,
Flathead not yet obtains breeding variety, and the germplasm such as cultured population is faced with decreased growth, premunition declines and genetic diversity reduces
The risk of degeneration.Therefore, carry out and the group germplasm of silver carp flathead is assessed, and breed improvement and selection and breeding work are carried out based on group's resource
Make, helps to reduce the dependence to wild resource, and provide safeguard for the sustainable development of its fishery cultivating.
The work development of Chinese Fishes genetic breeding is more, and has many successful cases, can to Chinese culture fishery health
Important support is played the role of in sustainable development.Wherein, unexpected mass incident is to carry out the simplest method of aquatic livestock breeding, is based primarily upon
Objective trait and phenotype selected by generation, relatively time saving and energy saving, can get very fast genetic progress in many environments, extensive
Applied to fish breeding；And in unexpected mass incident, the mating combination being usually directed between some populations between (or group) is related to.Silver carp and
Flathead is widely distributed in China, has more region population (or group), can provide substance base to carry out the research of its genetic improvement
Plinth.In addition, family selective breeding is also common breeding technique in fish genetic improvement, by establishing a large amount of familys, after grasping breeding
Family's coefficient and genealogical relationship in generation, the mating system be conducive in being selected by generation are formulated, can effectively reduce inbreeding probability and improve
Select pressure.However, the parent individual of silver carp flathead is big, conservation is of high cost, a large amount of familys of structure are relatively difficult, the oviposition of silver carp flathead in addition
Amount is big, and reproductive behavior basic data is deficient, the not yet reported development for having group or family selective breeding related work.
For above-mentioned practical problem, it is contemplated by the invention that being opened by microsatellite molecular marker (microsatellite)
Open up the researchs such as population genetic research and the paternity test of silver carp flathead.On the one hand, it will be appreciated that the germ plasm resource heredity of silver carp flathead is existing at present
Shape provides background data to carry out selection and breeding work；On the other hand, it by building paternity test technology, is carried to carry out family selective breeding
For technical support.In in the past few decades, also have to the genetic diversity Journal of Sex Research of the wild resource of Chinese Lian Yong groups more
Carry out, such as heredity based on morphology, isodynamic enzyme and molecular labeling etc. relatively, can provide basic ginseng to carry out unexpected mass incident
It examines；Wherein also there is the population genetic research carried out using microsatellite marker, but detection technique mostly uses polyacrylamide gel electricity
Swimming skills art exists in resolution ratio and accuracy and improves and improved space.And microsatellite marker is normal as the identification of animal pedigree
Molecular labeling, there is not been reported in the paternity test research of silver carp flathead；Therefore, it screens suitable microsatellite marker and is used for
Structure silver carp flathead paternity test technology has important practical significance.
In recent years, with the continuous development of molecular detection technology, the typing method of microsatellite marker allele is also got over
Come more accurate and efficient, by the detection method of traditional agarose gel electrophoresis and polyacrylamide gel electrophoresis, gradually sends out
Open up capillary electrophoresis detection technology；Wherein, in connected applications fluorophor (FAM and HEX etc.) Mdification primer and high-density DNA
The sequenator capillary electrophoresis detection technology of ginseng label (GS500 and ROX500 etc.) development is more and more to be used for microsatellite marker
Parting in, have many advantages, such as that high resolution, accuracy rate are high, reproducible and convenient for high-throughput detection.In addition, as biology tries
The continuous improvement of agent stability, multiple PCR technique are groped and build also increasingly to facilitate, and this method is also gradually applied to micro-
In the amplification of satellite markers, conventional efficient can be effectively improved and reduce experimental cost.
In summary the considerations of, the invention combination flathead genetic linkage maps pertinent literature information (Zhu et al., 2014)
And microsatellite sequence is downloaded from the websites NCBI, according to the target fragment standard in different length section, primer is redesigned, silver carp is utilized
Flathead individual DNA carries out PCR amplification, and by screen high amplification efficiency, Multiple Combination and primer concentration optimization and etc. grope structure
Multiplex PCR system is built, expanding effect is tested using 2.5% agarose gel electrophoresis.Finally, the ends sense primer 5' are carried out
Fluorophor modifies (FAM or HEX), and silver carp flathead individual DNA is recycled to carry out multiplexed PCR amplification, and product is complete certainly by ABI3730XL
Dynamic DNA sequencer carries out capillary electrophoresis detection, and the allele stripe size that each site corresponds on individual passes through
GeneMapper v4.0 softwares are read out, and can be ultimately utilized in Population variation research and paternity test analysis.
Invention content
The purpose of the present invention is overcoming above-mentioned shortcoming, a kind of microsatellite Multiplex fluorescent PCR side that silver carp flathead is general is provided
Method can be used for the researchs such as population genetic and the paternity test of two kinds of fishes.
Technical scheme of the present invention, a kind of method of the general microsatellite Multiplex fluorescent PCR of silver carp flathead collect silver carp flathead first
Fin ray sample, and genomic DNA is extracted using the method for traditional phenol-chloroform, then according to purpose expanding fragment length difference
It is required that being related to primer pair, amplified band clearly 3 group of 4 heavy PCR system is chosen, is related to 12 microsatellite markers altogether, finally to drawing
Object carries out fluorescent decoration and amplification verification.
Detailed process is as follows：
(1) sample collection of silver carp flathead and the preparation of DNA：Silver carp flathead fin ray sample is collected respectively, is stored in absolute ethyl alcohol；Using
The method of traditional phenol-chloroform extracts genomic DNA, and quality and purity, profit are detected by 1.0~1.5% agarose gel electrophoresis
Nucleic acid concentration is measured with UV detector, is finally diluted to 20~50ng/ μ L, and be stored in 0~4 DEG C.
(2) design of silver carp flathead micro-satellite primers and screening：Based on flathead genetic linkage maps documentation & info (Zhu et al.,
2014) Primer is utilized according to the requirement of purpose expanding fragment length difference to the related microsatellite sequence downloaded from NCBI
6.0 software Design primers of Premier, and biotech firm is sent to synthesize.The DNA that relevant primer is obtained by step (1) be template into
Row PCR amplification, amplified production are detected using 2.0~2.5% agarose gel electrophoresis, retain the good primer pair of expanding effect.
(3) silver carp flathead microsatellite multiplex PCR is groped and is optimized：It, first will amplification based on the primer pair that step (2) screening obtains
The carry out combination of two of band length difference carries out 2 heavy PCR amplifications, then the combination of 3 pairs and 4 pairs primers of progress is groped successively.Expand
Volume increase object is detected by 2.0~2.5% agarose gel electrophoresis, and adjusts each primer in multiplex PCR system according to expanding effect
To concentration.It is final to obtain amplified band clearly 3 group of 4 heavy PCR system, be related to 12 microsatellite markers altogether, each site and its
Primer information is as shown in table 1.Wherein, 4 microsatellite locus of multiplex PCR system 1 be respectively Arsd684, Hysd61-5,
Hysd9-2 and Arsd549；4 microsatellite locus of multiplex PCR system 2 are respectively Arsd380, Arsd443, Hysd3112-1
And Arsd639；4 microsatellite locus of multiplex PCR system 3 are respectively Hym435, Arsd86, Arsd291 and Arsd99.
(4) fluorescent decoration is carried out to primer and amplification is verified：The ends forward primer 5' in step (3) described site are carried out glimmering
Light base group modification (FAM or HEX), and using the DNA of step (1) acquisition as template, according to the 3 group 4 heavy PCR bodies of step (3) structure
System carries out PCR amplification, and amplified production carries out allelic gene typing after the detection of 2.5% agarose gel electrophoresis is qualified.
2.5% Ago-Gel testing result of 3 group of 4 general weight PCR system amplified production of silver carp flathead of the present invention is such as
Shown in Fig. 1.Wherein, the electrophoretic band for marking M is D2000 DNA Marker (TIANGEN), marks the electricity of 1-1,1-2 and 1-3
3 samples that band is multiplex PCR system 1 of swimming repeat, and the electrophoretic band of mark 2-2,2-2 and 2-3 are the 3 of multiplex PCR system 2
A sample repeats, and the electrophoretic band of mark 3-1,3-2 and 3-3 are 3 samples repetition of multiplex PCR system 3.
The parting of multiple PCR products described in step (4) is to use the full-automatic DNA sequencer capillaries of ABI3730XL
Electrophoresis detection, using GS500 as internal standard, the allele size that each site of individual is read by 4.0 softwares of GeneMapper v is believed
It ceases (bp), concrete outcome is as shown in Figure 2.
The figure respectively show system 1, system 2 and system 34 weight PCR systems each site amplified band size as a result,
Abscissa is based on the stripe size (bp) for being designated as reference in GS500, and ordinate is the identification intensity of fluorescent decoration signal, from figure
In it can be seen that the amplified fragments of adjacent sites in each system there are larger intervals.
In the general microsatellite Multiplex fluorescent PCR method of above-mentioned silver carp flathead：The purpose of design primer described in step (2)
Fragment length difference refers to that 4 different sections of 100~180bp, 180~260bp, 260~340bp and 340~420bp are long
Degree, avoids the amplified band of adjacent sites from being overlapped as possible.
The PCR amplification system of step (2) the micro-satellite primers screening includes 2 × Taq MasterMix (CWBIO), 10 μ
L, DNA profiling (20~50ng/ μ L) 1 μ L and upstream and downstream primer (10 μM) each 0.5 μ L, are used in combination ddH2O is supplemented to 20 μ L.
Micro-satellite primers described in step (2) screening pcr amplification reaction program be：94 DEG C of pre-degeneration 3min；Then 94
DEG C denaturation 30s, 55 DEG C of renaturation 30s, 72 DEG C of extension 40s, totally 35 cycles；Last 72 DEG C of extensions 10min, 4 DEG C of preservations.
12 microsatellite locus and primer information involved by step (3) 3 group of 4 weight PCR system are as shown in table 1.
3 group of 4 weight PCR system described in step (3) and step (4) include 2 × Taq MasterMix (CWBIO), 12.5 μ L,
2.2~2.8 μ L (dosage is as shown in table 1) of DNA profiling (50ng/ μ L) 2 μ L and primer mixed liquor, use ddH2O is supplemented to 25 μ L.
The PCR programs used in multi-PRC reaction program described in step (3) and step (4) and step (2) primer screening
It is identical.
Step (4) the fluorescent decoration method is that the site adjacent to purpose length in 4 sites of multiplex PCR system uses
Different fluorophors are modified (FAM and HEX, as shown in table 1), convenient for differentiating the amplification item of adjacent sites using different colours
Band.
The microsatellite locus and primer information table of 1 silver carp flathead of table, 3 group of 4 weight PCR system
Note:F indicates that forward primer, R indicate reverse primer；Fluorescent decoration label is held in forward primer 5 '.
Beneficial effects of the present invention：The advantage of the invention is that 12 microsatellite locus involved by the invention are suitable for silver carp
With the DNA cloning of two kinds of fishes of flathead, its application range can be effectively improved；And it is described 3 group 4 weight PCR system can effectively reduce experiment and
Testing cost improves working efficiency.In addition, invention has been evaded adjacent based on expanding fragment length difference and fluorescent decoration difference
The overlapping of site amplified band and obscure；And capillary electrophoresis detection is carried out using the full-automatic DNA sequencers of ABI3730XL, it can
The accuracy, accuracy and repeatability for effectively improving detection are conducive to extensive and mass experimental implementation.The invention institute
The method of stating can provide reliable technical support for correlative studys such as silver carp flathead population genetic and paternity tests.
Description of the drawings
Fig. 1 is the 2.5% Ago-Gel detection knot of 3 group of 4 general weight PCR system amplified production of silver carp flathead of the present invention
Fruit.
Fig. 2 is that the general 3 group 4 heavy PCR system amplified production of silver carp flathead of the present invention passes through GeneMapper v4.0 softwares
Reading result.
Fig. 3 is the principal coordinate analysis figure of 4 groups of silver carp flathead in embodiment 1.
Fig. 4 is the principal coordinate analysis figure of silver carp flathead 180 tail individual in embodiment 1.
Fig. 5 difference microsatellites are counted and paternity test success rate correspondence figure.
Specific implementation mode
The specific implementation mode of the present invention is described in detail below in conjunction with specific example.
Embodiment 1 is for the Population variation analysis of silver carp and flathead based on microsatellite marker
(1) silver carp and each 2 groups of flathead are collected respectively in THE LOWER YANGTZE VALLEY, 45 tail sample of each group amounts to 4 groups 180
Individual, and extracting genome DNA is carried out using phenol-chloroform method, integrality is detected through 1% agarose gel electrophoresis, utilization is ultraviolet
Spectrophotometer carries out Concentration Testing, and is diluted to 50ng/ μ L, is stored in 0 DEG C.
(2) DNA based on total 180 individual of 4 Ge Lian flatheads groups, utilizes 4 heavy PCR of 3 groups of fluorescent decorations of the present invention
System carries out PCR amplification.
(3) complete using ABI3730XL after 2.5% agarose gel electrophoresis qualification to pcr amplification product random sampling
Automated DNA sequenator carries out capillary electrophoresis detection, is based on GS500 internal standard references, is carried out using GeneMapper v4.0 softwares
Amplified band length is read out.
(4) it utilizes Excel tables to arrange the Allelotype data of corresponding 12 microsatellite locus of each individual, and converts phase
Format is answered, number of alleles (K), apparent heterozygosity (H of each site of 3.0 software statistics of CERVUS in silver carp and flathead are utilizedO)、
It is expected that heterozygosity (HE) and the genetic parameters such as polymorphism information content (PIC), the results are shown in Table 2；And it is soft using GENALEX 6.5
Part carries out principal coordinate analysis (Principal coordinates based on the genetic distance between 4 Ge Lian flatheads groups and 180 tail individuals
Analysis, PCoA), as a result as shown in Figure 3 and Figure 4.
According to the above analysis result, it can be seen that 12 microsatellite locus described in the invention, it can be effective for the group of silver carp flathead
The researchs such as body hereditary variation and Germplasm Identification.
The genetic diversity parametric statistics of table 2 silver carp flathead, 12 general microsatellite locus
Embodiment 2 is used for the paternity test to flathead artificial propagation parent and filial generation and analyzes
(1) the 12 male and ripe individuals of 12 female flatheads are utilized, according to partial factors Mating design (Partial factor
Mating design, Gjedrem 2005) carry out artificial propagation, it is divided into 4 mating groups, every group 3, to parent, is combined interior 1 tail
Raun (or milter) carries out artificial insemination with 2 tail milters (or raun).Fertilized eggs mixing hatching, and carry out the fry training that routinizes
It educates.
(2) sample collection of parent and filial generation, 24 tail parent's fin ray sample of clip, utilization are anhydrous during artificial propagation
Preservation is fixed in ethyl alcohol；And 192 odd amount in addition to the round numbers are acquired for the full fish of summer flower fry (~30 age in days), it is fixed using absolute ethyl alcohol
It preserves.
(3) extracting genome DNA is carried out for sample to above-mentioned 24 tail parent and 192 odd amount in addition to the round numbers using phenol-chloroform method, through 1%
Agarose gel electrophoresis detects integrality, carries out Concentration Testing using ultraviolet specrophotometer, and be diluted to 50ng/ μ L, preserves
In 0 DEG C.
(4) DNA based on above-mentioned parent and offspring individual, using invent 4 weight PCR systems of 3 groups of fluorescent decorations into
Row PCR amplification.
(5) complete using ABI3730XL after 2.5% agarose gel electrophoresis qualification to pcr amplification product random sampling
Automated DNA sequenator carries out capillary electrophoresis detection, is based on GS500 internal standard references, is carried out using GeneMapper v4.0 softwares
Amplified band length is read out.
(6) Allelotype data that Excel tables arrange parent and offspring individual corresponds to 12 microsatellite locus is utilized, and
Corresponding format is converted, paternity test analysis is carried out using 3.0 softwares of CERVUS.Qualification result is shown as shown in table 3, to 186 tails
Individual obtains precise Identification (confidence level 95%), identifies that success rate is 96.88%, identifies that successfully individual genealogical relationship meets people
The Mating design scheme of work breeding, rate of accuracy reached 100%.As shown in figure 5, according to the analysis of different loci number, find to use 7
The above site, so that it may to reach higher identification success rate (90% or so or more).Therefore, the invention 3 group of 4 heavy PCR
System method disclosure satisfy that the needs built to pedigree in the work of silver carp flathead genetic breeding.
The paternity test result (confidence level 95%) of 3 flathead parent of table and filial generation
So, the above is simply to illustrate that the features of the present invention, and not limitation of the present invention, related technical field
Those of ordinary skill according to the variation that the present invention makes in corresponding technical field should belong to the present invention protection category.
Sequence table
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<120>A kind of method of the general microsatellite Multiplex fluorescent PCR of silver carp flathead
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Claims (9)
1. a kind of method of the general microsatellite Multiplex fluorescent PCR of silver carp flathead, it is characterized in that：Silver carp flathead fin ray sample is collected first,
And genomic DNA is extracted using the method for traditional phenol-chloroform, then it is related to drawing according to the requirement of purpose expanding fragment length difference
Object pair chooses amplified band clearly 3 group of 4 heavy PCR system, is related to 12 microsatellite markers altogether, finally carries out fluorescence to primer
Modification and amplification verification.
2. the method for the general microsatellite Multiplex fluorescent PCR of silver carp flathead described in claim 1, it is characterized in that 3 group of 4 heavy PCR system
It is specific as follows：
System 1：Including 4 microsatellite locus, respectively Arsd684, Hysd61-5, Hysd9-2 and Arsd549；System 2：Packet
Include 4 microsatellite locus, respectively Arsd380, Arsd443, Hysd3112-1 and Arsd639；System 3：Including 4 microsatellites
Site, respectively Hym435, Arsd86, Arsd291 and Arsd99.
3. the method for the general microsatellite Multiplex fluorescent PCR of silver carp flathead described in claim 1, it is characterized in that 3 group of 4 heavy PCR system
The primer pair nucleotide sequence of middle use is specific as follows：
System 1：
The sites Arsd684 forward primer is as shown in SEQ ID NO.1, the sites Arsd684 reverse primer such as SEQ ID NO.2 institutes
Show；
The sites Hysd61-5 forward primer is as shown in SEQ ID NO.3, the sites Hysd61-5 reverse primer such as SEQ ID NO.4 institutes
Show；
The sites Hysd9-2 forward primer is as shown in SEQ ID NO.5, the sites Hysd9-2 reverse primer such as SEQ ID NO.6 institutes
Show；
The sites Arsd549 forward primer is as shown in SEQ ID NO.7, the sites Arsd549 reverse primer such as SEQ ID NO.8 institutes
Show；
System 2：
The sites Arsd380 forward primer is as shown in SEQ ID NO.9, the sites Arsd380 reverse primer such as SEQ ID NO.10 institutes
Show；
The sites Arsd443 forward primer is as shown in SEQ ID NO.11, the sites Arsd549 reverse primer such as SEQ ID NO.12 institutes
Show；
The sites Hysd3112-1 forward primer is as shown in SEQ ID NO.13, the sites Hysd3112-1 reverse primer such as SEQ ID
Shown in NO.14；
The sites Arsd639 forward primer is as shown in SEQ ID NO.15, the sites Arsd639 reverse primer such as SEQ ID NO.16 institutes
Show；
System 3：
The sites Hym435 forward primer is as shown in SEQ ID NO.17, the sites Hym435 reverse primer such as SEQ ID NO.18 institutes
Show；
The sites Arsd86 forward primer is as shown in SEQ ID NO.19, the sites Arsd86 reverse primer such as SEQ ID NO.20 institutes
Show；
The sites Arsd291 forward primer is as shown in SEQ ID NO.21, the sites Arsd291 reverse primer such as SEQ ID NO.22 institutes
Show；
The sites Arsd99 forward primer is as shown in SEQ ID NO.23, the sites Arsd99 reverse primer such as SEQ ID NO.24 institutes
Show.
4. the method for the general microsatellite Multiplex fluorescent PCR of silver carp flathead as described in claim 1, it is characterized in that being as follows：
（1）The sample collection of silver carp flathead and the preparation of DNA：Silver carp flathead fin ray sample is collected respectively, is stored in absolute ethyl alcohol；Using phenol-
The method of chloroform extracts genomic DNA, detects quality and purity by a concentration of 1.0% ~ 1.5% agarose gel electrophoresis, utilizes
UV detector measure nucleic acid concentration, be finally diluted to 20 ~ 50ng/ μ L, and be stored in 0 ~ 4 DEG C it is spare；It is long-term to keep needing
It freezes in -18 ~ -20 DEG C；
（2）Fluorescent decoration and PCR amplification：It is glimmering that the ends forward primer 5' in the primer pair described in claim 3 carry out FAM or HEX
Light base group modification, and with step（1）Acquisition DNA is template, and PCR expansions are carried out according to 3 group of 4 weight PCR system described in claim 2
Increase, amplified production carries out allelic gene typing after the detection of a concentration of 2.0% ~ 2.5% agarose gel electrophoresis is qualified.
5. the method for the general microsatellite Multiplex fluorescent PCR of silver carp flathead as claimed in claim 4, it is characterized in that：When the PCR amplification,
To avoid the amplified band of adjacent sites from being overlapped, purpose expanding fragment length be divided into 100 ~ 180bp, 180 ~ 260bp, 260 ~
The different siding-to-siding block lengths of 4 of 340bp and 340 ~ 420bp.
6. the method for the general microsatellite Multiplex fluorescent PCR of silver carp flathead as claimed in claim 4, it is characterized in that：3 group of 4 weight PCR expands
Increasing system is 25 μ L systems, every group of 1 ~ 2 μ L of DNA profiling comprising 2 × Taq MasterMix, 12.5 μ L, 20 ~ 50ng/ μ L
With 2.2 ~ 2.8 μ L of primer mixed liquor, ddH is used2O is supplemented to 25 μ L.
7. the method for the general microsatellite Multiplex fluorescent PCR of silver carp flathead as claimed in claim 4, it is characterized in that：The micro-satellite primers
The pcr amplification reaction program of screening is：94 DEG C of pre-degeneration 3min；Then 94 DEG C of denaturation 30s, 55 DEG C of renaturation 30s, 72 DEG C extend
40s, totally 35 ~ 38 recycle；Last 72 DEG C of extensions 10min, 4 DEG C of preservations.
8. the method for the general microsatellite Multiplex fluorescent PCR of silver carp flathead as claimed in claim 6, it is characterized in that the primer mixed liquor is every
Group system is 2.2 ~ 2.8 μ L, and concrete composition is as follows：
System 1：10 μM of 0.4 μ L, Arsd684 reverse primer of Arsd684 forward primers, 10 μM of 0.4 μ L；Hysd61-5 forward directions are drawn
10 μM of 0.4 μ L, Hysd61-5 reverse primer of object, 10 μM of 0.4 μ L；10 μM of 0.3 μ L, Hysd9-2 of Hysd9-2 forward primers are reversed
10 μM of 0.3 μ L of primer；10 μM of 0.3 μ L, Arsd549 reverse primer of Arsd549 forward primers, 10 μM of 0.3 μ L；
System 2：10 μM of 0.4 μ L, Arsd380 reverse primer of Arsd380 forward primers, 10 μM of 0.4 μ L；Arsd443 forward directions are drawn
10 μM of 0.2 μ L, Arsd443 reverse primer of object, 10 μM of 0.2 μ L；Hysd3112-1 forward primers 10 μM of 0.3 μ L, Hysd3112-
1 10 μM 0.3 of reverse primer μ L；10 μM of 0.2 μ L, Arsd639 reverse primer of Arsd639 forward primers, 10 μM of 0.2 μ L；
System 3：10 μM of 0.4 μ L, Hym435 reverse primer of Hym435 forward primers, 10 μM of 0.4 μ L；Arsd86 forward primers 10
μM 0.4 10 μM of 0.4 μ L of μ L, Arsd86 reverse primer；10 μM of 0.2 μ L, Arsd291 reverse primer 10 of Arsd291 forward primers
μM 0.2μL；10 μM of 0.4 μ L, Arsd99 reverse primer of Arsd99 forward primers, 10 μM of 0.4 μ L.
9. the method for the general microsatellite Multiplex fluorescent PCR of silver carp flathead as claimed in claim 4, it is characterized in that：The fluorescent decoration
Method is specially：
System 1：The ends Arsd684 forward primer 5' are modified for FAM, and the ends Hysd61-5 forward primer 5' are modified for HEX, Hysd9-2
The ends forward primer 5' are modified for FAM, and the ends Arsd549 forward primer 5' are modified for HEX；
System 2：The ends Arsd380 forward primer 5' are modified for FAM, and the ends Arsd443 forward primer 5' are modified for HEX, Hysd3112-
1 ends forward primer 5' are modified for FAM, and the ends Arsd639 forward primer 5' are modified for HEX；
System 3：The ends Hym435 forward primer 5' are modified for FAM, and the ends Arsd86 forward primer 5' are modified for HEX, and Arsd291 is positive
The ends primer 5' are modified for FAM, and the ends Arsd99 forward primer 5' are modified for HEX.
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| CN113215272A (en) * | 2021-05-07 | 2021-08-06 | 西安理工大学 | Silver carp mark release and release effect evaluation method |
| CN113621710A (en) * | 2021-07-13 | 2021-11-09 | 武汉中科瑞华生态科技股份有限公司 | Bighead microsatellite marker primer and Bighead marker discharge effect evaluation method |
| CN113621711A (en) * | 2021-07-13 | 2021-11-09 | 武汉中科瑞华生态科技股份有限公司 | Dual PCR (polymerase chain reaction) microsatellite primer for genetic diversity analysis of bighead carp and application of dual PCR microsatellite primer |
| CN117165695A (en) * | 2023-09-12 | 2023-12-05 | 中国水产科学研究院长江水产研究所 | Introgression gene, primer, method and application for identifying Changfeng silver carp |
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| CN113215272A (en) * | 2021-05-07 | 2021-08-06 | 西安理工大学 | Silver carp mark release and release effect evaluation method |
| CN113215272B (en) * | 2021-05-07 | 2022-06-17 | 西安理工大学 | Silver carp mark release and release effect evaluation method |
| CN113621710A (en) * | 2021-07-13 | 2021-11-09 | 武汉中科瑞华生态科技股份有限公司 | Bighead microsatellite marker primer and Bighead marker discharge effect evaluation method |
| CN113621711A (en) * | 2021-07-13 | 2021-11-09 | 武汉中科瑞华生态科技股份有限公司 | Dual PCR (polymerase chain reaction) microsatellite primer for genetic diversity analysis of bighead carp and application of dual PCR microsatellite primer |
| CN113621711B (en) * | 2021-07-13 | 2023-09-22 | 武汉中科瑞华生态科技股份有限公司 | Dual PCR microsatellite primer for bighead genetic diversity analysis and application |
| CN113621710B (en) * | 2021-07-13 | 2023-11-03 | 武汉中科瑞华生态科技股份有限公司 | Bighead microsatellite marked primer and bighead mark releasing effect evaluation method |
| CN117165695A (en) * | 2023-09-12 | 2023-12-05 | 中国水产科学研究院长江水产研究所 | Introgression gene, primer, method and application for identifying Changfeng silver carp |
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