CN107641656A - A kind of snakehead single nucleotide polymorphism and application thereof - Google Patents
A kind of snakehead single nucleotide polymorphism and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of snakehead SNP (Single Nucleotide Polymorphism, SNP) mark and application thereof, the design of primers designed by the present invention, PCR amplifications and SSCP technologies can quickly, low cost, accurate inspection snakehead SNP.SNP has a wide range of applications in research fields such as the genetic map construction of aquatic livestock, molecular mark, genetic diversity Journal of Sex Research and biological evolutions.The inventive method is a kind of SNP of examination on DNA level and detection snakehead method, obtains the high SNP of polymorphism and is advantageous to snakehead genetic diversity Journal of Sex Research, the protection of germ plasm resource, molecular breeding and other aquatic animal research.
Description
Technical field
The invention belongs to molecular genetics and molecular ecology field, is related to SNP molecular labeling and its answers
With.More particularly to snakehead SNP molecular labeling and application.
Background technology
SNP (Single Nucleotide Polymorphism, SNP) refers to certain in genomic dna sequence
The single nucleotide acid of individual specific site morph caused by sequence polymorphism, including the conversion of single base, transversion, insertion and
The forms such as missing.Theoretically, SNP is probably 3 or 4 equipotential polymorphisms, but actually the overwhelming majority is that two equipotentials are polymorphic
Property, the polymorphism of more allelic forms in addition is very rare, and usually said SNP is two equipotential polymorphisms.This variation
It is probably conversion (C-T, being then G-A on its complementary strand), it is also possible to transversion (C-A, G-T, C-G, A-T).Wherein change
Incidence is always apparently higher than other several variations, and the SNP with conversion form variation accounts for 2/3, the generation of other several variations
Probability is similar.
In genomic DNA, any base is likely to occur variation, therefore coverages of the SNP in genome is just
Compare extensively, be both possible in gene order, it is also possible on the non-coding sequence beyond gene.According to SNPs in genome
In position can be divided into code area SNPs (coding region SNPs), gene periphery SNPs (perigenic SNPs) and base
SNPs (intergenic SNPs) 3 classes because between.Most of SNPs are located at the noncoding region of genome, to protein without direct shadow
Ring, this kind of SNPs plays the role of critically important in being studied as genetic marker in population genetic and biological evolution.Minority point
SNPs of the cloth in gene coding region is referred to as cSNPs, and (coding SNPs, the change of any base are likely to cause to encode amino
Acid changes, and changes of its sequence can influence the biological function of change protein, thus cSNP science of heredity especially with disease
Have great importance in related research.
In recent years, people's screening is unknown or oneself knows that SNP technology and method have a lot, most common to have probe technique
(TaqMan), denaturing gradient gel electrophoresis (DGGE), single-strand conformation polymorphism (SSCP), restricted of PCR one
Segment length polymorphism (PCR-RFLP), direct sequencing etc., various methods have his own strong points, and are obtained in different analysis fields
It is widely applied.Taq enzyme activity influence is larger when the sensitiveness of wherein TaqMan probe technology is examined, needs to note when designing probe
Meaning FRET transmission efficiency and PCR amplification efficiencies, probe design cost are higher；SSCP is cumbersome, time-consuming, as a result easily causes
Erroneous judgement；PCR-RFLP is to produce or eliminate some restriction enzyme sites according to the mutation of base, so as to utilize restriction enzyme
Specificity, with one or more restricted interior enzyme effects in same DNA fragmentation, if restriction enzyme site has SNPs sites, digestion
Difference just occurs in the size and number of fragment, then carries out electrophoresis detection it may determine that whether having SNPs sites and base
The type of replacement.Shortcoming is that this method is only applicable to detect the SNPs at restriction enzyme site, for the SNPs beyond restriction enzyme site then
It can not detect.Direct sequencing is the quickest, direct, but it is higher that cost is sequenced.Sent out with the leap of Protocols in Molecular Biology
Exhibition, there is a series of high sensitivity, high-throughout methods of genotyping again in recent years, large sample and more SNPs positions can be met
The Genotyping requirement of point.
SNPs molecular labelings are in the structure genetic linkage mapses of aquatic livestock, association analysis, QTL positioning, systematic growth point
Analysis, population genetic variations, cultivar identification etc. research are with a wide range of applications.Foreign scholar Stickney etc. is using few
Oligonucleotide microarray technology, construct first SNPs genetic linkage maps of zebra fish (Danio rerio).Collection of illustrative plates contains
25 linkage groups, its total length are 3000cM, there is 1930 SNPs sites, equispaced 6.98cM on collection of illustrative plates.HSIN etc. is used
" ssalar0l " high density SNP matrixes are to 60 groups of core families, 622 Atlantic Ocean fresh fish Salmo sala:Carry out Genotyping simultaneously
Build high density genetic linkage mapses, collection of illustrative plates comprise more than 96K SNP site and 29 groups of chain colonies of salmon, every group of SNP site
It is in notable positive correlation with chromosome length (r=0.95).Domestic scholars Li etc. passes through 250K SNP Array Construction channel catfish
Letulurus Punetuu genetic maps, contain 54 342 SNP sites, total length 2505.4cM, and female recombinates with male
Rate is 1.7：1 coverage rate is 90%；Zhang Jianyong designs 800 groups of SNP primers, filters out 200 groups of structure Chinese prawns
(Fenneropenueus chanensas) parent's genetic linkage mapses, include 16 linkage groups, 180 marks, total length
899.3cM coverage rates are respectively 51.94% and 53.77%, examine Chinese prawn body weight, the long linkage of characters of body notable according to collection of illustrative plates
Property, it was found that two SNP sites related to body weight of C2 904-168, C1 2871-235.Urtzi etc. is to being derived from 26 geography
Totally 1 331 longfinned tunnies (Thunnus ululungu) carry out SNP site Genotyping for position, it is found that overfishing influences
Longfinned tunny population structure and genetic diversity.Gene is carried out to Mediterranean, the Atlantic Ocean, the Indian Ocean and Pacific colony
Homogeneous population defines in level, it is found that historic decline, its kind does not occur in North Atlantic Ocean longfinned tunny effective population size
Group's genetic diversity is not significantly affected with evolution potentiality by overfishing.Jiang Lihua using SNP technologies to Zhoushan, Zhangpu,
The large yellow croaker (Pseudoscauenu croceu) of four geographical population in Chengmai and Ningde carries out Analysis of Genetic Background, obtains 57
The good parting site of group and 10 genotype, wherein AT, GC are rare mutation type, can in large yellow croaker genetic diversity and
Utilized in population structure analysis.Therefore, genetic researches of the SNPs to aquatic livestock from now on, the assignment of genes gene mapping and molecule auxiliary are studied
Breeding has important scientific meaning.
There are many applications in people, mouse, drosophila, zebra fish isotype biology on SNP marker, still
Have not seen the research report about snakehead SNP marker.In recent years due to environmental pollution, hydraulic engineering, artificial fishing etc. because
Element causes wild snakehead population quantity to decline, and fishery resources are petered out, and urgent need takes measures to protect wild snakehead germ plasm resource.
Snakehead genetic map, population structure, genetic diversity are studied, is advantageous to recovery, the protection of germ plasm resource, promotes fishery resources can
Lasting development.
The content of the invention
The present invention screens snakehead SNPs polymorphism using " Illumina Hiseq " PCR sequencing PCR and PCR-SSCP methods, and
The high SNPs of polymorphism is subjected to polymorphism checking in 30 snakehead individuals, so as to accelerate snakehead genetic diversity, germplasm money
Source protection and the research utilized, fishery resources sustainable development will be promoted..It is an object of the present invention to provide a kind of snakehead
SNP marker and application thereof.
To achieve the above object, the technical solution used in the present invention：A kind of snakehead single nucleotide polymorphism, bag
Include：
The SNP that snakehead OaSNP1 the 1457th is C or A,
The SNP that snakehead OaSNP2 the 569th is A or C,
The SNP that snakehead OaSNP3 the 156th is T or A,
The SNP that snakehead OaSNP4 the 2747th is C or G,
The SNP that snakehead OaSNP5 the 403rd is C or T,
The SNP that snakehead OaSNP6 the 759th is A or T,
The SNP that snakehead OaSNP7 the 352nd is C or A,
The SNP that snakehead OaSNP8 the 316th is C or A,
The SNP that snakehead OaSNP9 the 743rd is A or C,
The SNP that snakehead OaSNP10 the 810th is T or A,
The SNP that snakehead OaSNP11 the 1306th is G or C,
The SNP that snakehead OaSNP12 the 453rd is T or C,
The SNP that snakehead OaSNP13 the 228th is T or A,
The SNP that snakehead OaSNP14 the 3089th is G or A,
The SNP that snakehead OaSNP15 the 216th is A or G,
The SNP that snakehead OaSNP16 the 812nd is C or T,
The SNP that snakehead OaSNP17 the 498th is T or C,
The SNP that snakehead OaSNP18 the 1833rd is G or C,
The SNP that snakehead OaSNP19 the 795th is A or G,
The SNP that snakehead OaSNP20 the 281st is T or C,
The SNP that snakehead OaSNP21 the 390th is A or G,
The SNP that snakehead OaSNP22 the 176th is T or G,
The SNP that snakehead OaSNP23 the 235th is G or A,
The SNP that snakehead OaSNP24 the 866th is A or C,
The SNP that snakehead OaSNP25 the 385th is A or T,
The SNP that snakehead OaSNP26 the 2534th is A or C,
The SNP that snakehead OaSNP27 the 2247th is A or C,
The SNP that snakehead OaSNP28 the 891st is A or C,
The SNP that snakehead OaSNP29 the 675th is G or T,
The SNP that snakehead OaSNP30 the 977th is A or T,
The SNP that snakehead OaSNP31 the 565th is T or C,
The SNP that snakehead OaSNP32 the 201st is A or C,
The SNP that snakehead OaSNP33 the 498th is C or T,
The SNP that snakehead OaSNP34 the 315th is A or G,
The SNP that snakehead OaSNP35 the 142nd is G or T,
The SNP that snakehead OaSNP36 the 915th is T or C.
The invention provides a kind of primer pair for being used to detect snakehead single nucleotide polymorphism described above, including
Primer pair OaSNP1~OaSNP36, the OaSNP1 are by SEQ ID NO:1 and SEQ ID NO:2 composition, the OaSNP2 by
SEQ ID NO:3 and SEQ ID NO:4 compositions, the OaSNP3 is by SEQ ID NO:5 and SEQ ID NO:6 compositions, it is described
OaSNP4 is by SEQ ID NO:7 and SEQ ID NO:8 compositions, the OaSN5 is by SEQ ID NO:9 and SEQ ID NO:10 groups
Into the OaSNP6 is by SEQ ID NO:11 and SEQ ID NO:12 compositions, the OaSNP7 is by SEQ ID NO:13 and SEQ
ID NO:14 compositions, the OaSNP8 is by SEQ ID NO:15 and SEQ ID NO:16 compositions, the OaSNP9 is by SEQ ID
NO:17 and SEQ ID NO:18 compositions, the OaSNP10 is by SEQ ID NO:19 and SEQ ID NO:20 compositions, it is described
OaSNP11 is by SEQ ID NO:21 and SEQ ID NO:22 compositions, the OaSNP12 is by SEQ ID NO:23 and SEQ ID NO:
24 compositions, the OaSNP13 is by SEQ ID NO:25 and SEQ ID NO:26 compositions, the OaSNP14 is by SEQ ID NO:27
With SEQ ID NO:28 compositions, the OaSNP15 is by SEQ ID NO:29 and SEQ ID NO:30 composition, the OaSNP16 by
SEQ ID NO:31 and SEQ ID NO:32 compositions, the OaSNP17 is by SEQ ID NO:33 and SEQ ID NO:34 compositions, institute
OaSNP18 is stated by SEQ ID NO:35 and SEQ ID NO:36 compositions, the OaSNP19 is by SEQ ID NO:37 and SEQ ID
NO:38 compositions, the OaSNP20 is by SEQ ID NO:39 and SEQ ID NO:40 compositions, the OaSNP21 is by SEQ ID NO:
41 and SEQ ID NO:42 compositions, the OaSNP22 is by SEQ ID NO:43 and SEQ ID NO:44 compositions, the OaSNP23
By SEQ ID NO:45 and SEQ ID NO:46 compositions, the OaSNP24 is by SEQ ID NO:47 and SEQ ID NO:48 compositions,
The OaSNP25 is by SEQ ID NO:49 and SEQ ID NO:50 compositions, the OaSNP26 is by SEQ ID NO:51 and SEQ ID
NO:52 compositions, the OaSNP27 is by SEQ ID NO:53 and SEQ ID NO:54 compositions, the OaSNP28 is by SEQ ID NO:
55 and SEQ ID NO:56 compositions, the OaSNP29 is by SEQ ID NO:57 and SEQ ID NO:58 compositions, the OaSNP30
By SEQ ID NO:59 and SEQ ID NO:60 compositions, the OaSNP31 is by SEQ ID NO:61 and SEQ ID NO:62 compositions,
The OaSNP32 is by SEQ ID NO:63 and SEQ ID NO:64 compositions, the OaSNP33 is by SEQ ID NO:65 and SEQ ID
NO:66 compositions, the OaSNP34 is by SEQ ID NO:67 and SEQ ID NO:68 compositions, the OaSNP35 is by SEQ ID NO:
69 and SEQ ID NO:70 compositions, the OaSNP36 is by SEQ ID NO:71 and SEQ ID NO:72 compositions.
The invention provides a kind of method for detecting snakehead single nucleotide polymorphism described above, bag uses
Primer pair described above.
The invention provides a kind of detection method of snakehead SNP, methods described is by snakehead to be measured
The detection of single nucleotide polymorphism described above is carried out, determines the genotype of the snakehead to be measured.
Preferably, methods described specifically includes following steps：
(1) using snakehead complete genome DNA to be measured as template, using the primer pair described in claim 2 as primer, performing PCR is entered
Amplification；
(2) to having the mark of positive band to carry out PCR-SSCP in step (1), the genotype of snakehead is determined.
Preferably, pcr amplification reaction program is in the step (1)：
98 DEG C of pre-degeneration 5min, 98 DEG C of denaturation 20s, 58 DEG C~62 DEG C annealing 30s, 72 DEG C of extension 30s, 25 circulate, and 72
DEG C extension 8min.
Preferably, PCR-SSCP programs in the step (2)：95 DEG C of PCR primer progress denaturation 10 minutes and immediately ice bath
Processing, the denaturation agent prescription used in the denaturation include deionized formamide 49m1,0.5M pH8.0 EDTA 1m1, bromophenol blue
0.l g, dimethylbenzene cyanines FF 0.1g.
Preferably, the mass concentration of polyacrylamide gel used in the step (2) is 10%.
The beneficial effects of the present invention are：The invention discloses a kind of snakehead SNP marker and application thereof, set by the present invention
The design of primers of meter, PCR amplifications and SSCP technologies can quickly, low cost, accurate inspection snakehead SNP.SNP
In research fields such as the genetic map construction of aquatic livestock, molecular mark, genetic diversity Journal of Sex Research and biological evolutions
Have a wide range of applications.The inventive method is a kind of SNP of examination on DNA level and detection snakehead method, is obtained polymorphic
The high SNP of property is advantageous to snakehead genetic diversity Journal of Sex Research, the protection of germ plasm resource, molecular breeding and other aquatic livestocks and ground
Study carefully.
Brief description of the drawings
Fig. 1 is snakehead total serum IgE agarose gel electrophoresis figure.
Fig. 2 is snakehead Oa1, Oa2 and Oa3 the detection figures of Agilent 2100.
Fig. 3 is snakehead Oa4, Oa5 and Oa6 the detection figures of Agilent 2100.
Fig. 4 is the polymorphic detection figures of snakehead OaSNP 5.
Fig. 5 is the polymorphic detection figures of snakehead OaSNP 6.
Embodiment
The present invention is by snakehead RNA extraction with transcript profile library construction, transcript profile sequencing with after assembling, snp analysis, obtaining
To the SNP marker of snakehead, based on obtained snakehead SNP marker, using snakehead complete genome DNA to be measured as template, with primer pair
OaSNP1~OaSNP36 is primer, enters performing PCR amplification.PCR primer carries out preliminary screening, tool by 3% agarose electrophoresis first
The mark for having clear band further carries out SSCP Genotypings by 10% polyacrylamide gel electrophoresis again, determines OaSNP1
~OaSNP36 nucleotide polymorphisms.The polymorphism inspection of 30 individuals is carried out for the SNP of discovery, and it is provided
Detection method so that it is fast that SNP turns into snakehead genetic diversity, genetic map construction, one kind of molecular mark research
Speed, the molecular labeling effectively, conveniently detected, foundation is provided for snakehead genetic diversity Journal of Sex Research, the protection of germ plasm resource and exploitation.
Using snakehead Oa1, Oa2, Oa3, Oa4, Oa5 and Oa6 muscle as sample, useKit
(Invitrogen) method on by specification carries out RNA extraction, is summarized as follows.
Organize in 50-100mg/mL TRIzo1 ratio adds corresponding TRIzoI, electronic homogenate, room temperature places 5min,
After it is fully cracked, in 4 DEG C, 12000rpm centrifuges 5min.
Supernatant is taken, adds chloroform in 200 μ L chloroforms/mL TRIzoI ratios, vibration mixes 15min, and room temperature places 15min
Afterwards, in 4 DEG C, 12000rpm centrifuges 15min.
Supernatant is taken, isopropanol is added in 500 μ L isopropanols/mL TRIzoI ratio, gently mixes, room temperature places 5-
After 10min, in 4 DEG C, 12000rpm centrifuges 10min.
Supernatant is abandoned, 75% ethanol is added in 75% ethanol of 1mL/mL TRIzoI ratio, gently vibrates centrifuge tube, suspend
Precipitation, in 4 DEG C, 8000g centrifuges 5min.
Supernatant is abandoned, is as far as possible sopped up remnants ethanol, 5-l0min is dried or be dried in vacuo to room temperature.
The RNA of extraction is carried out using Illumina RNA purification kits (Illumina, San Diego, CA) pure
Change, method is according to operation instruction.
Prepare before RNA detections, by sample after melting on ice, fully mix and centrifuge, take 500ng samples to be detected.
Detection parameters, Ago-Gel concentration：2% agarose gel, voltage：5V/cm, time：15min
Testing result is shown in Fig. 1.
Carried RNA concentration and purity are detected using Nanodrop2000, agarose gel electrophoresis detection RNA is complete
Whole property, Agilent2100 measure RIN values, such as Fig. 2 and Fig. 3.Single requirement for construction data base RNA total amount 1ug, concentration >=50ng/ μ L,
OD260/280 is between 1.8~2.2, OD260/230 >=2.0, RIN >=8.0,28S:18S≥1.0.
Eukaryote mRNA 3' ends have the structure of ployA tails, are entered using the magnetic bead with Oligo (dT) with ployA
Row A-T base pairings, can isolate mRNA from total serum IgE, and group information is transcribed for analyzing.
Illumina platforms are sequenced for short sequence fragment, and the mRNA for being enriched with to obtain is complete RNA sequence, are put down
Equal length reaches several kb, it is therefore desirable to which it is interrupted at random.Fragmentation buffer are added, can be random by mRNA
Fragment into 300bp or so small fragment.
In the presence of reverse transcriptase, using random primer, one chain cDNA of synthesis is inverted by template of mRNA, is then carried out
Two chains synthesize, and form stable duplex structure.
The cDNA structures of double-strand are cohesive end, add End Repair Mix and are mended into flat end, then at 3 ' ends
End adds an A base, for connecting the joint of Y-shaped.
Machine is sequenced on Illumina Hiseq.Library is enriched with, and PCR expands 15 cycles；2% agarose gel reclaims purpose
Band (Certified Low Range Ultra Agarose)；TBS380 (Picogreen) is quantitative, is mixed by ratio data
Upper machine；Bridge-type PCR amplifications are carried out on cBot, generate clusters；Illumina Hiseq are sequenced.
Determined by TBS380 quantification, there is the c DNA fragmentations of double ends in Illumina Hi Seq 4000 (2
× 150bp reading length) it is sequenced on platform.Using Seq Prep (https://github.com/jstjohn/Seq
) and Sickle (https Prep://github.com/najoshi/sickle) software progress sequence removal of impurities.It is soft with Trinity
Part to impurity elimination post-fragment carry out sequence assembling and splicing (http://trinityrnaseq.sourceforge.net/)。
By the sequence spliced as a comparison template by software Samtools (http:// samtools.sourceforge.net/) and VarScan v.2.2.7 (http://varscan.sourceforge.net/)
Oa1, Oa2, Oa3, Oa4, Oa5 and Oa6 sequence information obtained are contrasted to obtain the information of candidate SNP respectively with template.
According to analysis result, primer5.0 software Design primers, 60 in Oa1, Oa2, Oa3, Oa4, Oa5 and Oa6 sample are randomly choosed
Individual SNP marker designs primer, is verified.Primer synthesis is completed by Shanghai Sheng Gong companies.Carried out using agarose gel electrophoresis
SNP primers amplification success rate is identified, it is found that 57 go out product to (95%) primer energy Successful amplification.In order to eliminate false positive interference,
By PCR-SSCP methods, 57 pairs of primers progress polymorphism checking to Successful amplification in 30 individuals altogether (is repeated twice altogether
Experiment), it is final to obtain 36 (63.1%) polymorphic SNP marker.Described primer pair OaSNP1~OaSNP36 is as follows：
Pcr amplification reaction program is：
98 DEG C of pre-degeneration 5min, 98 DEG C of denaturation 20s, 58 DEG C~62 DEG C annealing 30s, 72 DEG C of extension 30s, 25 circulate, and 72
DEG C extension 8min.
The reaction system of PCR amplifications is 50 μ L, comprising 1 μ L DNA profilings, 30 μ 2 × PCR of L Mix (CW2296, century health
For), upstream and downstream primer each 2 μ L, 15 μ L H2O。
The mass concentration of Ago-Gel is 3%.
PCR-SSCP is 95 DEG C of PCR primer progress denaturation 10 minutes and ice bath is handled immediately, and SSCP denaturation agent prescriptions include
Deionized formamide 49m1,0.5M EDTA (pH8.0) 1m1, bromophenol blue 0.l g, dimethylbenzene cyanines FF 0.1g.
The mass concentration of polyacrylamide gel is that 10%, OaSNP 2 and OaSNP 4 electrophoretogram is shown in Fig. 4 and Fig. 5.
Marker gives birth to the pBR322/Msp I DNA Marker of the bright Science and Technology Ltd. of KeYu using Beijing.
The invention discloses the examination of snakehead SNP and detection method, by design of primers, PCR amplifications and
SSCP technologies can quickly, low cost, accurately examine its SNP.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected
The limitation of scope is protected, although being explained in detail with reference to preferred embodiment to the present invention, one of ordinary skill in the art should
Understand, technical scheme can be modified or equivalent substitution, without departing from the essence of technical solution of the present invention
And scope.
Claims (8)
- A kind of 1. snakehead single nucleotide polymorphism, it is characterised in that including：The SNP that snakehead OaSNP1 the 1457th is C or A,The SNP that snakehead OaSNP2 the 569th is A or C,The SNP that snakehead OaSNP3 the 156th is T or A,The SNP that snakehead OaSNP4 the 2747th is C or G,The SNP that snakehead OaSNP5 the 403rd is C or T,The SNP that snakehead OaSNP6 the 759th is A or T,The SNP that snakehead OaSNP7 the 352nd is C or A,The SNP that snakehead OaSNP8 the 316th is C or A,The SNP that snakehead OaSNP9 the 743rd is A or C,The SNP that snakehead OaSNP10 the 810th is T or A,The SNP that snakehead OaSNP11 the 1306th is G or C,The SNP that snakehead OaSNP12 the 453rd is T or C,The SNP that snakehead OaSNP13 the 228th is T or A,The SNP that snakehead OaSNP14 the 3089th is G or A,The SNP that snakehead OaSNP15 the 216th is A or G,The SNP that snakehead OaSNP16 the 812nd is C or T,The SNP that snakehead OaSNP17 the 498th is T or C,The SNP that snakehead OaSNP18 the 1833rd is G or C,The SNP that snakehead OaSNP19 the 795th is A or G,The SNP that snakehead OaSNP20 the 281st is T or C,The SNP that snakehead OaSNP21 the 390th is A or G,The SNP that snakehead OaSNP22 the 176th is T or G,The SNP that snakehead OaSNP23 the 235th is G or A,The SNP that snakehead OaSNP24 the 866th is A or C,The SNP that snakehead OaSNP25 the 385th is A or T,The SNP that snakehead OaSNP26 the 2534th is A or C,The SNP that snakehead OaSNP27 the 2247th is A or C,The SNP that snakehead OaSNP28 the 891st is A or C,The SNP that snakehead OaSNP29 the 675th is G or T,The SNP that snakehead OaSNP30 the 977th is A or T,The SNP that snakehead OaSNP31 the 565th is T or C,The SNP that snakehead OaSNP32 the 201st is A or C,The SNP that snakehead OaSNP33 the 498th is C or T,The SNP that snakehead OaSNP34 the 315th is A or G,The SNP that snakehead OaSNP35 the 142nd is G or T,The SNP that snakehead OaSNP36 the 915th is T or C.
- A kind of 2. primer pair of snakehead single nucleotide polymorphism for described in test right requirement 1, it is characterised in that bag Primer pair OaSNP1~OaSNP36, the OaSNP1 are included by SEQ ID NO:1 and SEQ ID NO:2 composition, the OaSNP2 by SEQ ID NO:3 and SEQ ID NO:4 compositions, the OaSNP3 is by SEQ ID NO:5 and SEQ ID NO:6 compositions, it is described OaSNP4 is by SEQ ID NO:7 and SEQ ID NO:8 compositions, the OaSN5 is by SEQ ID NO:9 and SEQ ID NO:10 groups Into the OaSNP6 is by SEQ ID NO:11 and SEQ ID NO:12 compositions, the OaSNP7 is by SEQ ID NO:13 and SEQ ID NO:14 compositions, the OaSNP8 is by SEQ ID NO:15 and SEQ ID NO:16 compositions, the OaSNP9 is by SEQ ID NO:17 and SEQ ID NO:18 compositions, the OaSNP10 is by SEQ ID NO:19 and SEQ ID NO:20 compositions, it is described OaSNP11 is by SEQ ID NO:21 and SEQ ID NO:22 compositions, the OaSNP12 is by SEQ ID NO:23 and SEQ ID NO: 24 compositions, the OaSNP13 is by SEQ ID NO:25 and SEQ ID NO:26 compositions, the OaSNP14 is by SEQ ID NO:27 With SEQ ID NO:28 compositions, the OaSNP15 is by SEQ ID NO:29 and SEQ ID NO:30 composition, the OaSNP16 by SEQ ID NO:31 and SEQ ID NO:32 compositions, the OaSNP17 is by SEQ ID NO:33 and SEQ ID NO:34 compositions, institute OaSNP18 is stated by SEQ ID NO:35 and SEQ ID NO:36 compositions, the OaSNP19 is by SEQ ID NO:37 and SEQ ID NO:38 compositions, the OaSNP20 is by SEQ ID NO:39 and SEQ ID NO:40 compositions, the OaSNP21 is by SEQ ID NO: 41 and SEQ ID NO:42 compositions, the OaSNP22 is by SEQ ID NO:43 and SEQ ID NO:44 compositions, the OaSNP23 By SEQ ID NO:45 and SEQ ID NO:46 compositions, the OaSNP24 is by SEQ ID NO:47 and SEQ ID NO:48 compositions, The OaSNP25 is by SEQ ID NO:49 and SEQ ID NO:50 compositions, the OaSNP26 is by SEQ ID NO:51 and SEQ ID NO:52 compositions, the OaSNP27 is by SEQ ID NO:53 and SEQ ID NO:54 compositions, the OaSNP28 is by SEQ ID NO: 55 and SEQ ID NO:56 compositions, the OaSNP29 is by SEQ ID NO:57 and SEQ ID NO:58 compositions, the OaSNP30 By SEQ ID NO:59 and SEQ ID NO:60 compositions, the OaSNP31 is by SEQ ID NO:61 and SEQ ID NO:62 compositions, The OaSNP32 is by SEQ ID NO:63 and SEQ ID NO:64 compositions, the OaSNP33 is by SEQ ID NO:65 and SEQ ID NO:66 compositions, the OaSNP34 is by SEQ ID NO:67 and SEQ ID NO:68 compositions, the OaSNP35 is by SEQ ID NO: 69 and SEQ ID NO:70 compositions, the OaSNP36 is by SEQ ID NO:71 and SEQ ID NO:72 compositions.
- 3. a kind of method of snakehead single nucleotide polymorphism for described in test right requirement 1, it is characterised in that including Primer pair described in usage right requirement 2.
- 4. a kind of detection method of snakehead SNP, it is characterised in that methods described is by entering to snakehead to be measured The detection of single nucleotide polymorphism described in row claim 1, determine the genotype of the snakehead to be measured.
- 5. detection method according to claim 4, it is characterised in that methods described specifically includes following steps：(1) using snakehead complete genome DNA to be measured as template, using the primer pair described in claim 2 as primer, performing PCR amplification is entered；(2) to having the mark of positive band to carry out PCR-SSCP in step (1), the genotype of snakehead is determined.
- 6. detection method according to claim 5, it is characterised in that pcr amplification reaction program is in the step (1)：98 DEG C of pre-degeneration 5min, 98 DEG C of denaturation 20s, 58 DEG C~62 DEG C annealing 30s, 72 DEG C of extension 30s, 25 circulations, 72 DEG C are prolonged Stretch 8min.
- 7. detection method according to claim 5, it is characterised in that PCR-SSCP programs in the step (2)：PCR is produced 95 DEG C of thing progress is denatured 10 minutes and ice bath is handled immediately, and the denaturation agent prescription used in the denaturation includes deionized formamide 49m1,0.5M pH8.0 EDTA 1m1, bromophenol blue 0.l g, dimethylbenzene cyanines FF 0.1g.
- 8. detection method according to claim 5, it is characterised in that polyacrylamide used coagulates in the step (2) The mass concentration of glue is 10%.
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Citations (2)
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| CN102912017A (en) * | 2012-09-29 | 2013-02-06 | 华东师范大学 | Method for quickly and accurately identifying northern snakehead, Taiwan snakehead and hybrid snakehead |
| CN107217099A (en) * | 2017-06-28 | 2017-09-29 | 中国水产科学研究院珠江水产研究所 | A kind of SNP marker identified available for snakehead genetic sex and supermale fish and its application |
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| CN102912017A (en) * | 2012-09-29 | 2013-02-06 | 华东师范大学 | Method for quickly and accurately identifying northern snakehead, Taiwan snakehead and hybrid snakehead |
| CN107217099A (en) * | 2017-06-28 | 2017-09-29 | 中国水产科学研究院珠江水产研究所 | A kind of SNP marker identified available for snakehead genetic sex and supermale fish and its application |
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| Title |
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| MINGSONG XIAO等: "Development of 36 SNP markers in Ophiocephalus argus Cantor based on high-throughput sequencing", 《CONSERVATION GENET RESOUR》 * |
| 陈双雅等 主编: "《鱼类分子生物学物种鉴定技术》", 30 September 2012, 厦门大学出版社 * |
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