CN106048027A - Grouper-growth-rate-related SNP (single-nucleotide polymorphism) marker and application thereof - Google Patents
Grouper-growth-rate-related SNP (single-nucleotide polymorphism) marker and application thereof Download PDFInfo
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Abstract
The invention discloses an SNP (single-nucleotide polymorphism) marker and application thereof. The SNP marker is the 501st-site base A or T from the 5' terminal in the nucleotide sequence disclosed as SEQ ID NO:1. The SNP marker is closely related to Saddletail grouper growth rate, and can be effectively used for molecular marker assisted breeding of Saddletail groupers.
Description
Technical field
The present invention relates to SNP marker and application thereof.In particular it relates to what the Epinephelus coioides speed of growth was correlated with
SNP marker, for detect the primer of aforementioned SNP marker to and test kit, aforementioned SNP marker, primer to, test kit at angled tape stone
Purposes in speckle fish selection-breeding, and the method for the detection Epinephelus coioides speed of growth.
Background technology
Cabrilla is famous and precious marine fish.The most during the last ten years, cabrilla artificial breeding technology relative maturity, stone
The cultivation scale of speckle fish the most gradually expands, developing rapidly of cabrilla industry, for promoting fisherman's increased income, meets national aquatic products
Demand, optimizes culture fishery structure significant.But, to plant matter and degenerate, it is that cabrilla industry can be held that improved seeds lack
The continuous Main Bottleneck developed in a healthy way.Therefore, cultivate cabrilla improved Varieties and become the key of China's cabrilla industry development.
Traditional fish breeding method places one's entire reliance upon phenotype, there is cycle length and the efficiency obstacle that cannot go beyond such as low.
Molecular breeding, i.e. molecular marker assisted selection breeding, refer to utilize DNA molecular marker that breeding material is selected, comprehensively change
The important economical trait of good selection-breeding species, is the breeding method of traditional genetic breeding and modern molecular biology combination.Point
Sub-breeding is that fish breeding opens up a new way, and along with the development of modern biotechnology, molecular marker is at fish breeding
In effect will become increasingly conspicuous.In the breeding of cabrilla, it is desirable to by closely related for growth traits, and with number
The selection of the amount closely linked DNA marker of character, to realize seed selection in early days and to improve the target of breeding accuracy, thus obtains more
Big genetic progress.
But, the molecular marker that present stage can be effective to the growth traits of cabrilla breeding relevant still has to be excavated.
Summary of the invention
It is contemplated that at least solve one of technical problem present in prior art.To this end, one object of the present invention
It is to propose one relevant to cabrilla growth traits, it is possible to be effective to the SNP marker of cabrilla selection-breeding.
Wherein it is desired to explanation, (single nucleotide polymorphism, SNP, i.e. mononucleotide is many for SNP
State property) it is that the molecule proposed by the human genome research center scholar Lander of Massachusetts Institute Technology for 1996 is lost
Pass labelling, be primarily referred to as in genomic level the DNA sequence polymorphism caused by the variation by single core thuja acid.SNP shows
Polymorphism relate only to the variation of single base, performance is to have conversion, transversion, insert and disappearance etc..
According to an aspect of the present invention, the invention provides the SNP marker that a kind of Epinephelus coioides speed of growth is relevant.
According to embodiments of the invention, this SNP marker is nucleotide sequence (total length 1001bp) shown in SEQ ID NO:1 from 5 ' ends the
501 bit bases represent with Y, and Y represents A or T.According to embodiments of the invention, nucleotide sequence shown in SEQ ID NO:1 is as follows:
ATGGAATTGACCCTCATTCGTCAAGCCATCACATGTTAACATCAGACTTTTAAATGAACATATGTGCGTCCTACTGC
CAAACCTGAGACTTACTAATTCCATCTTTGTCTTTCAATCAATCTCACATCTGCTTGGAAAACACGTTTATTTTTTA
GATCCGCAGTATATATTTGAATTATGCATAGAAAAGTTCAGGAAAAAACTTTTTTACGTCTATCGTCGTGCTTTATA
GTTAAGAATAACAACATATTCATTGTTTCTATTGATTAAAAATGTAACATTTTATATTGAATATCTAAAATTTCCTA
CTTCCATACGCAAGAAATCCAAAAACGATAAATACTGGGTGCTTGACAGAAGAAGGAATACAGAAATACACAGGTGT
GTCTCCTTATCTTGTTCATGTCTGATGAAGGGCTTGTGCCCGAAACGTCACATGGGGTGATTAGGGATGTTCCTGGC
CACTAATTTCCCTGTCGACTAAACAAACATTTTAATTAYGACTAGCTGACTAGTCTTAAAAAAAGGAAAACACTCAG
AAACAAGTCAAAATTTAGCACATTGTCCTAAAAAATATTGTGTGCATTAAGAGCCCTTTGAAGCCTGTCAGTCAAAA
GTTAACCACTAAAATAACATCTAAAATAAATAAATCGCCTCTGACATGTGGGGCACATTGAACCTTGCAGATTATCC
GACAGAAATGATACCAATTCACTTTAAAGTTAACAAAACAACCTATAATAACATCTCAAAAAAGAAAAATTGCTGCT
GAATTCATTTTCTAGATCAAAATACTGCCAAACCGTGCTGGTTTTGTGAGACGACATCTCTCCGATTTCCCACTGTG
CTGTTTGAAAACTCCAGTCTACTCAAGCATTACTGTGGGGAGGGGGACTGCTGTCTGATGGCTCTATAGCACCCGCT
AGTGGCAGGTGGCCACATGACAGTTAGCCTTGTACAGGAAGAAAACACTCATTTGTTTTACCGACTAATATTTCTGG
(SEQ ID NO:1).
Inventor finds, this site genotype is that the isozygoty body weight of Epinephelus coioides of TT or heterozygosis AT is significantly higher than
Genotype is the Epinephelus coioides of AA of isozygotying herein.And then, according to embodiments of the invention, by detecting the upper of Epinephelus coioides
State SNP, it is possible to effectively determine its speed of growth, specifically, as it was previously stated, this SNP site is isozygoty TT or heterozygosis AT
It is the Epinephelus coioides of AA of isozygotying that the body weight of Epinephelus coioides is significantly higher than genotype herein, the genotype of such as this SNP site
During for TT or AT, then can determine the individuality belonging to fast growth of Epinephelus coioides to be measured.Thus, inventor determines,
The SNP marker of the present invention is closely related with the speed of growth of Epinephelus coioides, it is possible to be effective to the molecule mark of Epinephelus coioides
Note assistant breeding.And then can carry out selecting in early days according to actual breeding demand growth selection speed epinephelus coioides breeding material
Select, be further able to be effectively improved the efficiency of breeding and accuracy, improve the genetic level of Epinephelus coioides reproductive population, thus
Epinephelus coioides improved seeds can be selected accurately and efficiently.Additionally, according to some embodiments of the present invention, utilize this
Bright SNP marker carries out Epinephelus coioides molecular mark, has early screening, time-consuming, with low cost, accurate
The advantage that property is high.
According to a further aspect in the invention, present invention also offers a kind of SNP for detecting the foregoing present invention
The primer pair of labelling.According to embodiments of the invention, described primer is to having the nucleotide sequence shown in SEQ ID NO:2-3.
Specifically, the sequence of the primer pair of the present invention is as follows:
Forward primer: 5 '-GGGTGCTTGACAGAAGAAGG-3 ' (SEQ ID NO:2)；
Downstream primer: 5 '-GCTTCAAAGGGCTCTTAATGC-3 ' (SEQ ID NO:3).
According to embodiments of the invention, the primer of the present invention is utilized to survey the above-mentioned of Epinephelus coioides to can effectively treat
The fragment at the SNP marker place relevant to the speed of growth carries out PCR amplification, and then can effectively be realized this SNP by order-checking
The detection of labelling, determines the genotype in this SNP marker site of Epinephelus coioides to be measured, and then can effectively determine angled tape stone to be measured
The speed of growth of speckle fish.Specifically, this SNP marker site genotype is the life of Epinephelus coioides of TT or heterozygosis AT of isozygotying
It is the Epinephelus coioides of AA of isozygotying that long speed is significantly higher than genotype herein, such as when the genotype of this SNP site is TT or AT,
Then can determine the individuality belonging to fast growth of Epinephelus coioides to be measured.Thus, it is used for detecting the foregoing present invention
The primer pair of SNP marker, it is possible to be effective to the molecular mark of Epinephelus coioides, and then can assist the most real
Existing short time, low cost, high accuracy ground selection-breeding Epinephelus coioides improved seeds.
According to another aspect of the invention, present invention also offers a kind of examination for detecting foregoing SNP marker
Agent box.According to embodiments of the invention, this test kit comprises: be described previously for detecting the primer of the SNP marker of the present invention
Right.The test kit of the present invention i.e. comprises the primer pair with the nucleotide sequence shown in SEQ ID NO:2-3.According to the present invention
Embodiment, utilize the primer pair included in the test kit of the present invention, it is possible to effectively realize treating and survey the upper of Epinephelus coioides
State the polymorphic detection of the SNP marker relevant to the speed of growth, determine the gene in this SNP marker site of Epinephelus coioides to be measured
Type, and then can effectively determine the speed of growth of Epinephelus coioides to be measured.Specifically, this SNP marker site genotype is pure
It is the Epinephelus coioides of AA of isozygotying that the speed of growth of the Epinephelus coioides closing TT or heterozygosis AT is significantly higher than genotype herein, example
When being TT or AT such as the genotype of this SNP site, then can determine that Epinephelus coioides to be measured belongs to the individuality of fast growth.
Thus, the test kit of the SNP marker for detecting the foregoing present invention of the present invention, it is possible to be effective to Epinephelus coioides
Molecular mark, and then can assist and realize in early days short time, low cost, high accuracy ground selection-breeding Epinephelus coioides
Improved seeds.
In accordance with a further aspect of the present invention, present invention also offers the SNP marker of the foregoing present invention, primer to or
Test kit, the purposes in Epinephelus coioides selection-breeding.As it was previously stated, by can be used in detection the present invention's and Epinephelus coioides
The most aforesaid primer of reagent of the SNP marker that the speed of growth is relevant to or comprise the test kit etc. of this primer pair, it is possible to effectively
Ground detection determines the genotype of the above-mentioned SNP marker of Epinephelus coioides to be measured, and then can be the most true based on the genotype obtained
The speed of growth of fixed Epinephelus coioides to be measured such that it is able to effectively auxiliary Epinephelus coioides selection-breeding.
And then, according to a further aspect in the invention, present invention also offers a kind of Epinephelus coioides speed of growth of detecting
Method.According to embodiments of the invention, the method carries out the inspection of foregoing SNP marker by treating survey Epinephelus coioides
Survey, determine the speed of growth of described Epinephelus coioides to be measured.Specifically, can by can be used in detection the present invention's and angled tape
The most aforesaid primer of reagent of the SNP marker that the cabrilla speed of growth is relevant to or comprise the test kit etc. of this primer pair, right
Epinephelus coioides to be measured carries out PCR amplification, order-checking, in order to detection determines the gene of the above-mentioned SNP marker of Epinephelus coioides to be measured
Type, and then the speed of growth of Epinephelus coioides to be measured can be effectively determined based on the genotype obtained.Wherein, as it was previously stated, be somebody's turn to do
SNP marker site genotype is that the isozygoty speed of growth of Epinephelus coioides of TT or heterozygosis AT is significantly higher than genotype herein
For the Epinephelus coioides of the AA that isozygotys, such as when the genotype of this SNP site is TT, Epinephelus coioides the most to be measured belong to growth
Fireballing individuality.Thus, the method for the detection Epinephelus coioides speed of growth of the present invention, it is possible to quickly, examine efficiently and accurately
Survey the Epinephelus coioides speed of growth, and then the molecular mark of Epinephelus coioides can be effective to such that it is able to be auxiliary
Help and realize short time, low cost, high accuracy ground selection-breeding Epinephelus coioides improved seeds in early days.
It addition, the method for the detection Epinephelus coioides speed of growth according to the above embodiment of the present invention can also have as follows
Additional technical characteristic:
According to embodiments of the invention, treat and survey Epinephelus coioides and carry out the method for SNP marker detection and be not particularly limited.
Order-checking, single strand conformation polymorphism polymerase chain reaction (PCR single strand conformation
Polymorphism, PCR-SSCP), restriction fragment length polymorphism polymerase chain reaction (PCR-restriTCion
Fragment length polymorphism, PCR-RFLP) and the technology such as flight time mass spectrum all can realize the inspection of SNP
Survey.Wherein, order-checking is that a kind of accuracy is the highest, motility strong, the detection technique that flux is big, the detection cycle is short.Only need to be in SNP position
The both sides design pair of primers of point, the product of amplification 200-1000bp, then the gene of SNP site can be directly detected by order-checking
Type.Thus, the present invention uses the method for order-checking to carry out SNP marker detection.According to some concrete examples of the present invention, by treating
Survey Epinephelus coioides and carry out the detection of foregoing SNP marker, determine the speed of growth of described Epinephelus coioides to be measured, enter one
Step includes: extract the genomic DNA of Epinephelus coioides to be measured；Utilize foregoing primer pair, by described angled tape lithosporic to be measured
The genomic DNA of fish carries out PCR amplification, in order to obtain pcr amplification product；Described pcr amplification product is checked order, in order to obtain
Obtain sequencing result；Based on described sequencing result, determine the genotype of the described SNP marker of described Epinephelus coioides to be measured；And
The genotype of described SNP marker based on described Epinephelus coioides to be measured, determines the speed of growth of described Epinephelus coioides to be measured.
Thereby, it is possible to be effectively improved the efficiency of the detection Epinephelus coioides speed of growth.
According to embodiments of the invention, the method for the genomic DNA extracting Epinephelus coioides to be measured is not particularly limited, can
To use any of genome DNA extracting method or test kit to carry out.According to some concrete examples of the present invention, often use
Rule phenol chloroform method extracts the genomic DNA of Epinephelus coioides to be measured.Thereby, it is possible to effectively obtain the base that quality is good, purity is high
Because of group DNA, it is simple to subsequent step is carried out.
According to embodiments of the invention, the genomic DNA of described Epinephelus coioides to be measured is carried out the condition of PCR amplification not
It is particularly limited.According to some concrete examples of the present invention, the amplification system of this PCR amplification is calculated as with 25 μ l: 50-100ng/ μ l
The each 1 μ l of the upstream and downstream primer shown in SEQ ID NO:2-3, the dNTP of 10mmol/L of template DNA 1 μ l, 10pmol/ μ l
The Taq archaeal dna polymerase 0.125 μ l of mix 2.0 μ l, 5U/ μ l, 10 × PCR reaction buffer 2.5 μ l, surplus is distilled water；Should
PCR amplification reaction condition be: 94 DEG C 5 minutes；94 DEG C 30 seconds, 53 DEG C 30 seconds, 72 DEG C 30 seconds, 30 circulations；72 DEG C 5 minutes.
Thereby, it is possible to expand the fragment at the SNP marker place of the present invention quickly, efficiently and accurately, it is thus achieved that target amplification product, it is simple to
The carrying out of subsequent step.
According to embodiments of the invention, the method checking order described pcr amplification product is not particularly limited, if energy
Enough effectively acquisition pcr amplification product i.e. sequences of the fragment at SNP marker place.According to some concrete examples of the present invention,
Can use at least one selected from HISEQ2000, SOLiD, 454 and single-molecule sequencing method that described pcr amplification product is entered
Row order-checking.Thereby, it is possible to high flux, obtain sequencing result quickly, efficiently and accurately.
According to embodiments of the invention, based on sequencing result, by comparison cabrilla with reference to genome sequence, it is possible to effectively
The genotype determining the described SNP marker of Epinephelus coioides to be measured is TT, AA or AT.
According to embodiments of the invention, the speed of growth of TT or the AT genotype individuals of described SNP marker is significantly higher than AA
Genotype individuals.The i.e. foregoing SNP marker of the present invention is closely related with the speed of growth of Epinephelus coioides.Thus, based on
The genotype of this SNP marker of the Epinephelus coioides to be measured determined, it is possible to determine the life of Epinephelus coioides to be measured accurately and effectively
When the genotype of long speed i.e. growth and development traits, such as this SNP site is TT, belonging to of Epinephelus coioides the most to be measured grows speed
Spend fast individuality.And then the method for the present invention can be effective to the molecular mark of Epinephelus coioides such that it is able to
Auxiliary realizes short time, low cost, high accuracy ground selection-breeding Epinephelus coioides improved seeds in early days.
It should be noted that the SNP marker relevant to the Epinephelus coioides speed of growth of the present invention and application thereof have as
Lower advantage:
(1) SNP marker that the present invention provides is not limited by age of Epinephelus coioides, sex etc., can be used for angled tape lithosporic
The early stage selection-breeding of fish, can remarkably promote the breeding process of Epinephelus coioides；
(2) detection Epinephelus coioides as shown in SEQ ID NO.1 from 5 ' ends the method for the 501st SNP site, accurately may be used
Lean on, easy to operate；
(3) Epinephelus coioides detection of the SNP site of the 501st from 5 ' ends as shown in SEQ ID NO.1, for angled tape stone
The marker assisted selection of speckle fish growth traits provides scientific basis.
The additional aspect of the present invention and advantage will part be given in the following description, and part will become from the following description
Obtain substantially, or recognized by the practice of the present invention.
Detailed description of the invention
Unless specifically indicated, the general sense during term used herein has art of the present invention.
Below with reference to specific embodiment, the present invention will be described, it should be noted that these embodiments are only explanation
Property, and be not considered as limiting the invention.Unreceipted concrete technology or condition in embodiment, all according to normal experiment
Condition, such as Sambrook equimolecular Cloning: A Laboratory Manual (Sambrook J&Russell DW, Molecular cloning:a
Laboratory manual, 2001), or the condition according to manufacturer's description suggestion.Agents useful for same or the unreceipted life of instrument
Produce manufacturer person, be can by city available from conventional products.
The acquisition of the SNP marker that embodiment 1 is relevant to the Epinephelus coioides speed of growth
The acquisition of 1.1 Epinephelus coioides colonies
The colony used is the Epinephelus coioides of Grouper cultivating field, Hainan hatching on November 10th, 2010, Qin Benwei
Article 29, wild milter and 12 wild rauns (being trapped in the South China Sea in Sanya, Hainan), on December 10th, 2010,15,000
Tail fry is transferred to a net cage and continues to raise.On August 8th, 2011, select 198 individualities, clip fish at random from this net cage
Body dorsal rags is in 95% ethanol-20 DEG C preservation, for extracting genome DNA.
1.2 Epinephelus coioides extracting genome DNA
This test uses the genomic DNA in conventional phenol chloroform method extracting Epinephelus coioides fin ray, specifically comprises the following steps that
(1) take 0.3～0.5g fin ray in 1.5ml Eppendorf pipe, shred, superclean bench is uncapped dry
20min；
(2) after ethanol volatilizees substantially, TE buffer (10mmol/ml Tris, 1mmol/ml EDTA, SDS 5%, pH
=8.0) washing 1～2 time, adds 600 μ l DNA extract (0.001mol/L Tris-Cl, 0.1mol/L EDTA, SDS
5%, pH=8.0) and 3 μ l protease k (200mg/ml), 55 DEG C of water-bath digestion about 3h, front 30min every 10min jogs are centrifuged
Pipe 1 time, digests to liquid in pipe clarification；
(3) autogamy phenol chloroformic solution (phenol: chloroform: isoamyl alcohol=25:24:1) 600 μ l are added, the most reverse centrifugal
Pipe 10min, 12000r are centrifuged 10min.Take upper strata aqueous phase equal-volume above-mentioned phenol chloroform to extract again, until aqueous phase and organic facies it
Between there is no white precipitate；
(4) use chloroform 1 time again, take out supernatant, add the dehydrated alcohol precipitation DNA of 2 times of volume pre-coolings, reverse
Mixing, 4 DEG C stand 30min, 12000r and are centrifuged 10min, and precipitation use 70% washing with alcohol again, be centrifuged after drying precipitation add 50 μ l without
Bacterium water dissolution.4 DEG C save backup or-20 DEG C long-term preservations.
1.3 structure simplification gene order-checkings (Restriction site Associated DNA sequencing,
RAD-seq) SNP marker that library the acquisition Epinephelus coioides body weight that checks order are correlated with
Based on Hiseq2000 high-flux sequence platform, RAD is used to simplify gene order-checking method to 198 individual DNA
Sample checks order, and produces the data volume of about 0.4G, the average Epinephelus coioides genome covering 0.4X.The most also to this
198 individualities carry out the growth traits phenotypic evaluation such as body weight.PLINK software is used data to carry out processing Screening Treatment, then
Use EMMAX software based on mixed linear model to carry out GWAS analysis, from 261,366 SNP have found one and body weight
The SNP site of significant correlation.This SNP site is positioned at the 501bp site of sequence shown in SEQ ID NO.1, at SEQ ID NO.1
Sequence represents site at this with Y, and the base in site herein is A or C.This site genotype is TT or heterozygosis AT of isozygotying
The body weight of Epinephelus coioides to be significantly higher than genotype herein be the Epinephelus coioides of AA of isozygotying.
The sequence verification of the SNP marker that embodiment 2 is relevant to the Epinephelus coioides speed of growth and application
2.1 extract the genomic DNA in Epinephelus coioides fin ray to be measured
Epinephelus coioides to be measured, from the Epinephelus coioides colony in embodiment 1, randomly selects 180 tail fishes, according to enforcement
DNA extraction method extracting genomic DNA described in example 1.
2.2 amplifications nucleotide fragments containing SNP site
With the aforementioned genomic DNA of Epinephelus coioides each to be measured obtained that extracts as template, utilize forward primer F:5 '-
GGGTGCTTGACAGAAGAAGG-3 ' (SEQ ID NO:2) and reverse primer R:5 '-GCTTCAAAGGGCTCTTAATGC-3 '
(SEQ ID NO:3), amplifies the nucleotide fragments at SNP place to be measured.Wherein, PCR reaction system is calculated as with 25 μ l: 50-
100ng/ μ l template DNA 1 μ l, 10pmol/ μ l primers F and each 1 μ l of R, 10mmol/L dNTP mix 2.0 μ l, 5U/ μ l Taq
Archaeal dna polymerase 0.125 μ l, 10 × PCR reaction buffer 2.5 μ l, surplus is distilled water；PCR reaction condition is: 94 DEG C 5 minutes；
94 DEG C 30 seconds, 53 DEG C 30 seconds, 72 DEG C 30 seconds, 30 circulations；72 DEG C 5 minutes.
2.3 order-checkings identify SNP site genotype
The each pcr amplification product obtained in above-mentioned steps is carried out on ABI3730 sequenator unidirectional order-checking, identifies SEQ
The genotype of (i.e. the SNP marker of the present invention) at 501bp in ID NO:1 sequence.Wherein, 180 Epinephelus coioides to be measured are individual
Genotype and the body weight thereof of this SNP site are as shown in table 1 below.
The genotype of 1 180 these SNP site of individuality of table and body weight thereof
2.4 SNP site genotype and the association analysis of the speed of growth
Result based on table 1, utilizes SAS9.0 software Mixed program to carry out linear analogue and analyzes the genotype of SNP site
With the relatedness of the speed of growth, representing phenotypic number with whose body weight when wherein analyzing, the model of employing is as follows:
Yijk=μ+Gi+aj+eijk。
Wherein, YijkFor whose body weight value, μ colony body weight average, GiFor genotype effects vector, ajFor minor-polygene to
Amount, eijkFor random residual effect vector.
The genotype of SNP site see table 2 with the association analysis result of the speed of growth.
The genotypic frequency of table 2 SNP site and the association analysis with body weight
As shown in Table 2, TT homozygous whose body weight average is maximum, and AT heterozygous whose body weight average is taken second place, and AA is homozygous
Whose body weight average is minimum.
Association analysis result shown in table 2 shows, the average of AT genotype individuals body weight and AA genotype individuals body weight
The difference of average reaches pole significance level (P < 0.01), TT genotype individuals body weight average and AA genotype individuals body weight average
Difference reaches pole significance level (P < 0.01).And then, it was demonstrated that nucleotide sequence shown in SEQ ID NO:1 from 5 ' end the 501st
Base A or T, with Epinephelus coioides speed of growth significant correlation, the SNP marker being correlated with for the Epinephelus coioides speed of growth, this SNP
The speed of growth of the TT genotype individuals of labelling is significantly higher than AA genotype individuals, and the speed of growth of AT genotype individuals is significantly high
In AA genotype individuals.
In the description of this specification, reference term " embodiment ", " some embodiments ", " example ", " specifically show
Example " or the description of " some examples " etc. means to combine this embodiment or example describes specific features, structure, material or spy
Point is contained at least one embodiment or the example of the present invention.In this manual, to the schematic representation of above-mentioned term not
Necessarily refer to identical embodiment or example.And, the specific features of description, structure, material or feature can be any
One or more embodiments or example in combine in an appropriate manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that: not
These embodiments can be carried out multiple change in the case of departing from the principle of the present invention and objective, revise, replace and modification, this
The scope of invention is limited by claim and equivalent thereof.
Claims (8)
1. the SNP marker that an Epinephelus coioides speed of growth is relevant, it is characterised in that the sequence of described SNP marker such as SEQ
Shown in ID NO:1, the 501st bit base from 5 ' ends of sequence shown in described SEQ ID NO:1 is A or T.
SNP marker the most according to claim 1, it is characterised in that TT or the AT genotype individuals of described SNP marker
The speed of growth is significantly higher than AA genotype individuals.
3. the primer pair being used for test right requirement SNP marker described in 1 or 2, it is characterised in that described primer is to having
Nucleotide sequence shown in SEQ ID NO:2-3.
4. the test kit being used for test right requirement SNP marker described in 1 or 2, it is characterised in that comprise: claim 3
Described primer pair.
5. the SNP marker described in claim 1 or 2, the primer described in claim 3 to or claim 4 described in test kit
Purposes in Epinephelus coioides selection-breeding.
6. the method detecting the Epinephelus coioides speed of growth, it is characterised in that weigh by treating survey Epinephelus coioides
Profit requires the detection of the SNP marker described in 1 or 2, determines the speed of growth of described Epinephelus coioides to be measured.
Method the most according to claim 6, it is characterised in that carry out claim 1 or 2 by treating survey Epinephelus coioides
The detection of described SNP marker, determines the speed of growth of described Epinephelus coioides to be measured, farther includes:
Extract the genomic DNA of Epinephelus coioides to be measured；
Utilize the primer pair described in claim 3, the genomic DNA of described Epinephelus coioides to be measured carried out PCR amplification, in order to
Obtain pcr amplification product；
Described pcr amplification product is checked order, in order to obtain sequencing result；
Based on described sequencing result, determine the genotype of the described SNP marker of described Epinephelus coioides to be measured；And
The genotype of described SNP marker based on described Epinephelus coioides to be measured, determines the growth of described Epinephelus coioides to be measured
Speed.
Method the most according to claim 7, it is characterised in that the life of TT or the AT genotype individuals of described SNP marker
Long speed is significantly higher than AA genotype individuals.
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