CN107022608A - SNP marker and its application - Google Patents
SNP marker and its application Download PDFInfo
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- CN107022608A CN107022608A CN201710167375.0A CN201710167375A CN107022608A CN 107022608 A CN107022608 A CN 107022608A CN 201710167375 A CN201710167375 A CN 201710167375A CN 107022608 A CN107022608 A CN 107022608A
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Abstract
The invention discloses SNP marker and its application.Wherein, the SNP marker is SEQ ID NO：The 501st the bit base C or T from holding 5 ' of nucleotide sequence shown in 1.The SNP marker and the speeds of growth of Epinephelus coioides of the present invention is closely related, can effective for Epinephelus coioides molecular mark.
Description
Technical field
The present invention relates to SNP marker and its application.In particular it relates to related to the Epinephelus coioides speed of growth
SNP marker, primer pair and kit for detecting foregoing SNP marker, foregoing SNP marker, primer pair, kit are in angled tape stone
Purposes in spot fish seed selection, and the method for detecting the Epinephelus coioides speed of growth.
Background technology
Grouper is rare marine fish.Closely during the last ten years, grouper artificial breeding technology relative maturity, stone
The cultivation scale of spot fish also gradually expands, developing rapidly for grouper industry, for promoting fisherman's increased income, meets national aquatic products
Demand, optimization culture fishery structure is significant.But, germplasm is degenerated, and it is that grouper industry can be held that improved seeds, which lack,
The continuous main bottleneck developed in a healthy way.Therefore, cultivate grouper improved Varieties turns into the key of China's grouper industry development.
Traditional fish breeding method places one's entire reliance upon phenotype, there is cycle length and the low obstacle that can not go beyond of efficiency.
Molecular breeding, i.e. molecular marker assisted selection breeding refers to select breeding material using DNA molecular marker, and synthesis changes
The important economical trait of good seed selection species, is the breeding method that traditional genetic breeding is organically combined with modern molecular biology.Point
Sub- breeding opens up a new way for fish breeding, and with the development of modern biotechnology, molecular labeling is in fish breeding
In effect will become increasingly conspicuous.In the breeding of grouper, it is desirable to by closely related for growth traits, and with number
The selection of the DNA marker of character close linkage is measured, with the target realized early stage seed selection and improve breeding accuracy, so as to obtain more
Big genetic progress.
However, can still have to be excavated effective for the related molecular labeling of the growth traits of grouper breeding at this stage.
The content of the invention
It is contemplated that at least solving one of technical problem present in prior art.Therefore, it is an object of the invention to
Propose a kind of, SNP marker that can effective for grouper seed selection and its application related to grouper growth traits.
, wherein it is desired to explanation, (single nucleotide polymorphism, SNP, i.e. mononucleotide are more by SNP
State property) it is that the molecule that the human genome research center scholar Lander by Massachusetts Institute Technology in 1996 is proposed is lost
Mark is passed, is primarily referred to as in genomic level as the DNA sequence polymorphism caused by the variation of single nucleotide acid.SNP is shown
Polymorphism relate only to the variation of single base, performance is that have conversion, transversion, insertion and missing etc..
Another object of the present invention is to provide it is a kind of be used to detecting the related SNP marker of the Epinephelus coioides speed of growth,
Kit and its application.
A further object of the present invention is to provide a kind of method for detecting the Epinephelus coioides speed of growth.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of related SNP marker of Epinephelus coioides speed of growth, the sequence such as SEQ ID NO of the SNP marker：1 institute
Show, such as SEQ ID NO：The 501st bit base from holding 5 ' of sequence shown in 1 is C or T.Embodiments in accordance with the present invention, SNP marks
It is designated as SEQ ID NO：Nucleotide sequence (total length 1001bp) shown in 1 from 5 ' hold the 501st bit base represented with Y, Y represent C or
T。
The speed of growth of the TT genotype individuals of above-mentioned SNP marker is significantly higher than CC genotype individuals.Inventor has found, is somebody's turn to do
Genotype is that the body weight of homozygosis TT Epinephelus coioides is significantly higher than the Epinephelus coioides that genotype herein is homozygosis CC at site.
And then, embodiments in accordance with the present invention, by detecting the above-mentioned SNP of Epinephelus coioides, can effectively determine its speed of growth,
Specifically, as it was previously stated, it is homozygosis CC that the body weight that the SNP site is homozygosis TT Epinephelus coioides, which is significantly higher than genotype herein,
Epinephelus coioides, such as when the genotype of the SNP site is TT, then can determine Epinephelus coioides to be measured belongs to growth speed
The fast individual of degree.Thus, inventor determines, SNP marker of the invention and the speed of growth of Epinephelus coioides are closely related, can
Effective for the molecular mark of Epinephelus coioides.And then can be according to actual breeding demand growth selection speed to oblique
Band grouper breeding material carries out Seedling selection, is further able to effectively improve the efficiency and accuracy of breeding, improves angled tape stone
The genetic level of spot fish reproductive population, so as to accurately and efficiently select Epinephelus coioides improved seeds.In addition, according to
Some embodiments of the present invention, carry out Epinephelus coioides molecular mark, with early stage using the SNP marker of the present invention
The high advantage of screening, saving time, with low cost, accuracy.
Present invention also offers a kind of primer pair for being used to detect above-mentioned SNP marker.The primer pair has SEQ ID
NO：Nucleotide sequence shown in 2-3.Specifically, the sequence of primer pair of the invention is as follows：
Sense primer：5’-CTCCTCTGCTGCTCAGCTTT-3’(SEQ ID NO：2)；
Anti-sense primer：5’-CCGTACCTCCACTGATGTGA-3’(SEQ ID NO：3).
The above-mentioned SNP related to the speed of growth for surveying Epinephelus coioides can be effectively treated using the primer pair of the present invention
Fragment where mark enters performing PCR amplification, and then can effectively realize the detection to the SNP marker by sequencing, determines to be measured
The genotype in the Epinephelus coioides SNP marker site, and then can effectively determine the speed of growth of Epinephelus coioides to be measured.Specifically
It is pure that the speed of growth for the Epinephelus coioides that genotype is homozygosis TT, which is significantly higher than genotype herein, at ground, the SNP marker site
CC Epinephelus coioides are closed, such as when the genotype of the SNP site is TT or CT, then can determine that Epinephelus coioides to be measured belong to
The individual of fast growth.Thus, for detecting the primer pair of above-mentioned SNP marker, can effective for Epinephelus coioides point
Sub- marker-assisted breeding, so can aid in early stage realize the short time, low cost, high accuracy seed selection Epinephelus coioides it is excellent
Kind.
Present invention also offers a kind of kit for being used to detect above-mentioned SNP marker.Included in the kit just like SEQ
ID NO：The primer pair of nucleotide sequence shown in 2-3.Wrapped in embodiments in accordance with the present invention, the kit using the present invention
The primer pair contained, can effectively realize the polymorphism for treating the above-mentioned SNP marker related to the speed of growth for surveying Epinephelus coioides
Detection, determines the genotype in the Epinephelus coioides to be measured SNP marker site, and then can effectively determine Epinephelus coioides to be measured
The speed of growth.Specifically, genotype is significantly higher than this for the speed of growth of homozygosis TT Epinephelus coioides at the SNP marker site
Locate the Epinephelus coioides that genotype is homozygosis CC, such as when the genotype of the SNP site is TT, then can determine angled tape stone to be measured
Spot fish belongs to the individual of fast growth.Thus, the kit for being used to detect above-mentioned SNP marker of the invention, can be effective
In the molecular mark of Epinephelus coioides, and then early stage can be aided in realize short time, low cost, select high accuracy
Educate Epinephelus coioides improved seeds.
Present invention also offers the purposes of above-mentioned SNP marker, primer pair or kit in Epinephelus coioides seed selection.As before
Described, the reagent of the SNP marker related to the Epinephelus coioides speed of growth by can be used in the detection present invention is for example foregoing
Primer pair or kit comprising the primer pair etc., can effectively detect the above-mentioned SNP marks for determining Epinephelus coioides to be measured
The genotype of note, and then the genotype based on acquisition can effectively determine the speed of growth of Epinephelus coioides to be measured, so as to
Effectively auxiliary Epinephelus coioides seed selection.
Present invention also offers a kind of method for detecting the Epinephelus coioides speed of growth.This method is by treating deviational survey band stone
Spot fish carries out the detection of above-mentioned SNP marker, determines the speed of growth of the Epinephelus coioides to be measured.Specifically, energy can be passed through
It is enough in the reagent example primer pair as the aforementioned or bag of the SNP marker related to the Epinephelus coioides speed of growth of the detection present invention
Kit containing the primer pair etc., treats survey Epinephelus coioides and enters performing PCR amplification, sequencing, angled tape stone to be measured is determined to detect
The genotype of the above-mentioned SNP marker of spot fish, and then the genotype based on acquisition can effectively determine the life of Epinephelus coioides to be measured
Long speed.Wherein, as it was previously stated, genotype is notable for the speed of growth of homozygosis TT Epinephelus coioides at the SNP marker site
Higher than the Epinephelus coioides that genotype herein is homozygosis CC, such as when the genotype of the SNP site is TT, then angled tape stone to be measured
The individual for belonging to fast growth of spot fish.Thus, the method for the detection Epinephelus coioides speed of growth of the invention, can be fast
Speed, efficiently and accurately the detection Epinephelus coioides speed of growth, and then can be aided in effective for the molecular labeling of Epinephelus coioides
Breeding, so as to aid in early stage to realize short time, low cost, high accuracy ground seed selection Epinephelus coioides improved seeds.
In addition, it is according to the above embodiment of the present invention detection the Epinephelus coioides speed of growth method can also have it is as follows
Additional technical characteristic：
Embodiments in accordance with the present invention, the method for treating survey Epinephelus coioides progress SNP marker detection is not particularly limited.
Sequencing, single-strand conformation polymorphism PCR (PCR single strand conformation
Polymorphism, PCR-SSCP), RFLP PCR (PCR-restriTCion
Fragment length polymorphism, PCR-RFLP) and the technology such as flight time mass spectrum can realize SNP inspection
Survey.Wherein, sequencing is that a kind of accuracy highest, flexibility are strong, the detection technique that flux is big, detection cycle is short.Only need to be at SNP
The both sides design pair of primers of point, expands 200-1000bp product, then can directly detect the gene of SNP site by sequencing
Type.Thus, the present invention carries out SNP marker detection using the method for sequencing.According to some specific examples of the present invention, by treating
The detection that Epinephelus coioides carry out foregoing SNP marker is surveyed, the speed of growth of the Epinephelus coioides to be measured is determined, enters one
Step comprises the following steps：Extract the genomic DNA of Epinephelus coioides to be measured；, will be described to be measured using foregoing primer pair
The genomic DNA of Epinephelus coioides enters performing PCR amplification, to obtain pcr amplification product；The pcr amplification product is surveyed
Sequence, to obtain sequencing result；Based on the sequencing result, the base of the SNP marker of the Epinephelus coioides to be measured is determined
Because of type；And the genotype of the SNP marker based on the Epinephelus coioides to be measured, determine the Epinephelus coioides to be measured
The speed of growth.Thereby, it is possible to effectively improve the efficiency of the detection Epinephelus coioides speed of growth.
Embodiments in accordance with the present invention, the method for extracting the genomic DNA of Epinephelus coioides to be measured is not particularly limited, can
To be carried out using any of genome DNA extracting method or kit.According to some specific examples of the present invention, using normal
Advise the genomic DNA that phenol chloroform method extracts Epinephelus coioides to be measured.Thereby, it is possible to effectively obtain the base that quality is good, purity is high
Because of a group DNA, it is easy to subsequent step to carry out.
Embodiments in accordance with the present invention, the condition of performing PCR amplification is entered not by the genomic DNA of the Epinephelus coioides to be measured
It is particularly limited.According to some specific examples of the present invention, the amplification system of PCR amplifications is calculated as with 25 μ l：50-100ng/μl
μ l, the 10pmol/ μ l of template DNA 1 SEQ ID NO：The dNTP of upstream and downstream primer each 1 μ l, 10mmol/L shown in 2-3
μ l, the 5U/ μ l of the mix 2.0 μ l of 0.125 μ l, 10 × PCR reaction buffer of Taq archaeal dna polymerases 2.5, 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 circulation；72 DEG C 5 minutes.
The fragment where SNP marker thereby, it is possible to quickly, efficiently and accurately expand the present invention, obtains target amplification product, is easy to
The progress of subsequent step.
Embodiments in accordance with the present invention, are not particularly limited to the method that the pcr amplification product is sequenced, as long as energy
It is enough effectively to obtain the sequence that pcr amplification product is the fragment where SNP marker.According to some specific examples of the present invention,
It can use and the pcr amplification product is entered selected from HISEQ2000, SOLiD, 454 and at least one of single-molecule sequencing method
Row sequencing.Thereby, it is possible to high flux, it is quick, efficiently and accurately obtain sequencing result.
Embodiments in accordance with the present invention,, can be effective by comparing grouper reference gene group sequence based on sequencing result
The genotype for determining the SNP marker of Epinephelus coioides to be measured is TT or CC.
Embodiments in accordance with the present invention, the speed of growth of the TT genotype individuals of the SNP marker is significantly higher than CC genes
Type individual.Foregoing SNP marker i.e. of the invention and the speed of growth of Epinephelus coioides are closely related.Thus, based on determination
Epinephelus coioides to be measured the SNP marker genotype, can accurately and effectively determine Epinephelus coioides to be measured growth speed
Degree is growth and development traits, such as when the genotype of the SNP site is TT, then Epinephelus coioides to be measured belong to fast growth
Individual.And then the present invention method can effective for Epinephelus coioides molecular mark, so as to aid in
Early stage realizes short time, low cost, high accuracy ground seed selection Epinephelus coioides improved seeds.
It should be noted that the SNP marker related to the Epinephelus coioides speed of growth of the present invention and its application have such as
Lower advantage：
(1) limitation such as age, the sex of the SNP marker that the present invention is provided not by Epinephelus coioides, available for angled tape lithosporic
The early stage seed selection of fish, can remarkably promote the breeding process of Epinephelus coioides；
(2) method of detection Epinephelus coioides the 501st SNP site from holding 5 ' as shown in SEQ ID NO.1, accurately may be used
Lean on, it is easy to operate；
(3) detection of Epinephelus coioides SNP site of the 501st from holding 5 ' as shown in SEQ ID NO.1, is angled tape stone
The marker assisted selection of spot fish growth traits provides scientific basis.
The additional aspect and advantage of the present invention will be set forth in part in the description, and will partly become from the following description
Obtain substantially, or recognized by the practice of the present invention.
Embodiment
Unless specifically indicated, term used herein has the general sense in art of the present invention.
Below with reference to specific embodiment, the present invention will be described, it is necessary to which explanation, these embodiments are only explanation
Property, and be not considered as limiting the invention.Unreceipted particular technique or condition in embodiment, according to normal experiment
Condition, such as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J＆Russell DW, Molecular cloning:a
Laboratory manual, 2001), or according to the condition of manufacturer's specification suggestion.Agents useful for same or the unreceipted life of instrument
Manufacturer person is produced, being can be by the conventional products of acquisition purchased in market.
The acquisition of the SNP marker related to the Epinephelus coioides speed of growth of embodiment 1
The acquisition of 1.1 Epinephelus coioides colonies
The Epinephelus coioides of Hainan Grouper cultivating hatching on November 10th, 2010, Qin Benwei is in the colony used
29 wild milters and 12 wild rauns (South China Sea for being trapped in Sanya, Hainan), on December 10th, 2010,15,000 tails
Fry is transferred to a net cage and continues to raise.On August 8th, 2011,198 individuals, clip fish body are selected from the net cage at random
Dorsal rags are in -20 DEG C of preservations of 95% ethanol, for extracting genome DNA.
1.2 Epinephelus coioides extracting genome DNAs
This experiment is comprised the following steps that using the genomic DNA in conventional phenol chloroform method extracting Epinephelus coioides fin ray：
(1) take 0.3~0.5g fin rays in 1.5ml Eppendorf pipes, shred, drying of being uncapped on superclean bench
20min；
(2) after ethanol volatilizees substantially, TE buffer solutions (10mmol/ml Tris, 1mmol/ml EDTA, SDS 5%, pH
=8.0) wash 1~2 time, add 600 μ l DNA extracts (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-baths digestion 3h or so, preceding 30min is per the centrifugation of 10min jogs
Pipe 1 time, digestion to liquid in pipe clarification；
(3) autogamy phenol chloroformic solution (phenol is added：Chloroform：Isoamyl alcohol (V:V:V)=25：24：1) 600 μ l, gently run back and forth
The 10min of falling centrifuge tube, 12000r centrifuge 10min.Upper strata aqueous phase is taken to be extracted again with isometric above-mentioned phenol chloroform, until aqueous phase is with having
There is no white precipitate between machine phase；
(4) chloroform is used again 1 time, take out supernatant, add the absolute ethyl alcohol precipitation DNA of 2 times of volume precoolings, overturn
Mix, 4 DEG C standing 30min, 12000r centrifugation 10min, precipitation wash again with 70% ethanol, centrifugation dry precipitate after add 50 μ l without
Bacterium water dissolves.4 DEG C save backup or -20 DEG C long-term preservation.
1.3 build simplified gene order-checking (Restriction site Associated DNA sequencing, RAD-
Seq) library and it is sequenced and obtains the related SNP marker of Epinephelus coioides body weight
Based on Hiseq2000 high-flux sequence platforms, the gene order-checking method DNA individual to 198 is simplified using RAD
Sample is sequenced, and is produced 0.4G or so data volume, is averagely covered 0.4X Epinephelus coioides genome.Simultaneously also to this
198 individuals carry out the growth traits phenotypic evaluations such as body weight.Processing Screening Treatment is carried out to data using PLINK softwares, then
GWAS analyses are carried out using the EMMAX softwares based on mixed linear model, one and body weight are have found from 261,366 SNP
Significantly correlated SNP site.The SNP site is located at shown in SEQ ID NO.1 at the 501bp sites of sequence, in SEQ ID NO.1
Site at this is represented in sequence with Y, and the base in site is C or T herein.Genotype is homozygosis TT angled tape lithosporic at the site
The body weight of fish is significantly higher than the Epinephelus coioides that genotype herein is homozygosis CC.
The sequence verification of the SNP marker related to the Epinephelus coioides speed of growth of embodiment 2 and application
2.1 extract the genomic DNA in Epinephelus coioides fin ray to be measured
Epinephelus coioides colony of the Epinephelus coioides to be measured in embodiment 1, randomly selects 180 tail fishes, according to implementation
DNA extraction method extracting genomic DNA described in example 1.
2.2 nucleotide fragments of the amplification containing SNP site
Using the foregoing genomic DNA for extracting each Epinephelus coioides to be measured obtained as template, forward primer F is utilized：5’-
CTCCTCTGCTGCTCAGCTTT-3’(SEQ ID NO：2) with reverse primer R：5’-CCGTACCTCCACTGATGTGA-3’
(SEQ ID NO：3) nucleotide fragments where SNP marker to be measured, are amplified.Wherein, PCR reaction systems are calculated as with 25 μ l：
50-100ng/ μ l template DNAs 1 μ l, 10pmol/ μ l primers Fs and each μ l, 5U/ μ l of 1 μ l, 10mmol/L dNTP mix 2.0 of R
The μ l of 0.125 μ l, 10 × PCR reaction buffer of Taq archaeal dna polymerases 2.5, surplus is distilled water；PCR reaction conditions are：94℃5
Minute；94 DEG C 30 seconds, 53 DEG C 30 seconds, 72 DEG C 30 seconds, 30 circulation；72 DEG C 5 minutes.
2.3 sequencing identification SNP site genotype
By each pcr amplification product obtained in above-mentioned steps in being unidirectionally sequenced on ABI3730 sequenators, SEQ is recognized
ID NO：The genotype of (i.e. SNP marker of the invention) in 1 sequence at 501bp.Wherein, 60 Epinephelus coioides individuals to be measured should
The genotype and its body weight of SNP site are as shown in table 1 below.
The genotype and its body weight of 1 60, the table individual SNP site
| Individual numbering | Body weight (g) | Genotype | Individual numbering | Body weight (g) | Genotype |
| s127 | 20 | CC | s77 | 72 | CC |
| s39 | 32 | CC | s101 | 74 | CC |
| s115 | 36 | CC | s116 | 74 | CC |
| s119 | 36 | CC | s18 | 74 | CC |
| s75 | 36 | CC | s109 | 78 | CC |
| s8 | 36 | CC | s138 | 80 | CC |
| s155 | 38 | CC | s181 | 80 | CC |
| s22 | 40 | CC | s98 | 80 | CC |
| s66 | 40 | CC | s11 | 82 | CC |
| s64 | 42 | CC | s182 | 90 | CC |
| s68 | 46 | CC | s129 | 92 | CC |
| s90 | 46 | CC | s172 | 92 | CC |
| s13 | 48 | CC | s197 | 92 | CC |
| s144 | 48 | CC | s73 | 100 | CC |
| s34 | 48 | CC | s71 | 104 | CC |
| s4 | 48 | CC | s135 | 114 | CC |
| s62 | 48 | CC | s189 | 118 | CC |
| s143 | 50 | CC | s85 | 132 | CC |
| s20 | 50 | CC | s27 | 156 | CC |
| s156 | 52 | CC | s95 | 170 | CC |
| s102 | 54 | CC | s2 | 146 | TT |
| s6 | 54 | CC | s26 | 150 | TT |
| s23 | 56 | CC | s187 | 180 | TT |
| s142 | 58 | CC | s174 | 180 | TT |
| s175 | 64 | CC | s179 | 186 | TT |
| s91 | 64 | CC | s185 | 190 | TT |
| s123 | 66 | CC | s165 | 210 | TT |
| s147 | 68 | CC | s188 | 228 | TT |
| s69 | 70 | CC | s72 | 262 | TT |
| s152 | 72 | CC | s163 | 262 | TT |
The association analysis of 2.4SNP loci gene types and the speed of growth
Based on the result of table 1, the genotype that linear analogue analyzes SNP site is carried out using SAS9.0 software Mixed programs
With the relevance of the speed of growth, wherein representing phenotypic number during analysis with whose body weight, the model of use is as follows：
Yijk=μ+Gi+aj+eijk。
Wherein, YijkFor whose body weight value, μ colonies body weight average, GiFor genotype effects vector, ajFor minor-polygene to
Amount, eijkFor random residual effect vector.
The genotype of SNP site and the association analysis result of the speed of growth see the table below 2.
The genotype frequency of the SNP site of table 2 and the association analysis with body weight
As shown in Table 2, the homozygous whose body weight averages of the homozygous whose body weight average ratio CC of TT are big.
Association analysis result shown in table 2 shows, average and the TT genotype individuals body weight of GT genotype individuals body weight
The difference of average reaches pole significance (P<0.01).And then, it was demonstrated that SEQ ID NO：Nucleotide sequence shown in 1 is the from holding 5 '
501 bit base G or T, it is significantly correlated with the Epinephelus coioides speed of growth, it is the related SNP marker of the Epinephelus coioides speed of growth,
The speed of growth of the TT genotype individuals of the SNP marker is significantly higher than CC genotype individuals.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means to combine specific features, structure, material or the spy that the embodiment or example are described
Point is contained at least one embodiment of the present invention or example.In this manual, to the schematic representation of above-mentioned term not
Necessarily refer to identical embodiment or example.Moreover, specific features, structure, material or the feature of description 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
In the case of departing from the principle and objective of the present invention a variety of change, modification, replacement and modification can be carried out to these embodiments, this
The scope of invention is limited by claim and its equivalent.
SEQUENCE LISTING
<110>Zhenjiang Hua Da detection Co., Ltd
<120>SNP marker and its application
<130> 2017
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1001
<212> DNA
<213>Epinephelus coioides
<221>The nucleotide sequence of the related SNP marker of the Epinephelus coioides speed of growth
<400> 1
gcacgcactt taatagacat caaacatcga cggtgcgata tagccccagg gattatattg 60
cagcctgttg tgcgactgcc ggctgcaggg ctctcactca atactagatc agattcaaaa 120
atcgatttcc ccttcagtca cctaagtcgt tttcatatca ggtatgactg atttgtacca 180
tgtctgagta tgattgcccc cccacctcca ctcctctgct gctcagcttt cacatttaat 240
gctgttccgt agctaagtac actttcatca tcatctacct gacatttttg ctacgtgtag 300
aaaagggcgc cgcagacata cactgctgct tcagggacta tcatctgctt ctggccagag 360
gaggagctct ctaccaccca ctgaagctct gacagcaggg tggagattgc agcgaagcct 420
gatctaagtc tgtccacagt gggctgcgct gccatctgat atagagactc ttgcataacc 480
agttaaaaac atcttgtagt yacattaaat agcagtccag ctttgtgtta tcgaacagtt 540
gctgttcata cttgatgtgc actgtaccag aactttacac aagaccacct tttcaagtgg 600
acttgggcaa gcatacttcc ttgtacactg gtacactggt tcacactaat caaacaaagt 660
ggactttgag gtcaagtgta ctcagatctg ggcccaggtc tctgatgtga aacccccatt 720
agagacaaaa gaatgagaaa ataggttgaa aaatgccagt ttcccattaa ataatgcatt 780
tcctccgatg acaacacaag aaattaagga gtcatggttt aaaccataag ttagcaagta 840
attcctgtct gatttaaata gctaatcgaa cacacagcac tttcacatca gtggaggtac 900
ggtaatgtca tttcattcac tctgggcttc attctcaaag gctgtgtcat aaccagtttc 960
aaactagcgc tttaacacca actatgtatt aaacttcctt t 1001
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<221>Forward primer F
<400> 2
ctcctctgct gctcagcttt 20
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<221>Reverse primer R
<400> 3
ccgtacctcc actgatgtga 20
Claims (8)
1. a kind of related SNP marker of Epinephelus coioides speed of growth, it is characterised in that the sequence of the SNP marker such as SEQ
ID NO：Shown in 1, such as SEQ ID NO：The 501st bit base from holding 5 ' of sequence shown in 1 is C or T.
2. SNP marker according to claim 1, it is characterised in that the growth speed of the TT genotype individuals of the SNP marker
Degree is significantly higher than CC genotype individuals.
3. the primer pair of a kind of SNP marker for described in test right requirement 1 or 2, it is characterised in that the primer pair has
SEQ ID NO：Nucleotide sequence shown in 2-3.
4. the kit of a kind of SNP marker for described in test right requirement 1 or 2, it is characterised in that the kit includes power
Profit requires the primer pair described in 3.
5. the primer pair described in SNP marker, claim 3 described in claim 1 or 2 or the kit described in claim 4
Purposes in Epinephelus coioides seed selection.
6. a kind of method for detecting the Epinephelus coioides speed of growth, it is characterised in that weighed by treating survey Epinephelus coioides
Profit requires the detection of the SNP marker described in 1 or 2, determines the speed of growth of the Epinephelus coioides to be measured.
7. method according to claim 6, it is characterised in that this method specifically includes following steps：
(1) genomic DNA of Epinephelus coioides to be measured is extracted；
(2) using the primer pair described in claim 3, the genomic DNA of the Epinephelus coioides to be measured is entered into performing PCR amplification,
To obtain pcr amplification product；
(3) pcr amplification product is sequenced, to obtain sequencing result；
(4) sequencing result is based on, the genotype of the Epinephelus coioides SNP marker to be measured is determined；Deviational survey is treated based on described
Genotype with grouper SNP marker, determines the speed of growth of the Epinephelus coioides to be measured.
8. method according to claim 7, it is characterised in that the speed of growth of the TT genotype individuals of the SNP marker
It is significantly higher than CC genotype individuals.
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| CN112322759A (en) * | 2020-12-10 | 2021-02-05 | 镇江华大检测有限公司 | Detection method for identifying three kinds of cod based on high-throughput sequencing |
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