CN102653795B - Primers and method for distinguishing Thai marble goby fries from local marble goby fries - Google Patents
Primers and method for distinguishing Thai marble goby fries from local marble goby fries Download PDFInfo
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Abstract
The invention belongs to the field of DNA markers in the field of aquaculture science and in particular relates to primers and a method for distinguishing Thai marble goby (oxyeleotrismarmoratus) fries from local marble goby (odontobutispotamophila) fries by adopting microsatellite markers. A pair of primer sequences is shown in SEQIDNo.8 and SEQIDNo.9. The method comprises the following steps of: carrying out PCR (polymerase chain reaction) amplification on the samples to be distinguished by utilizing primers; and then detecting the PCR products by using 1% of agarose gel electrophoresis andcarrying out EB (ethidium bromide) staining, wherein the samples with 250bp stripes are Thai marble goby and the samples with 300bp stripes are local marble goby. The method has the following beneficial effects: the microsatellite markers obtained by the method can carry out stable amplification in oxyeleotrismarmoratus and odontobutispotamophila and have stable results and clear stripes; and themethod is simple and convenient to operate and is low in cost and strong in practicability.
Description
Technical field
The invention belongs to the application of Aquaculture Science field dna marker technology in production practice, be specifically related to use microsatellite marker to Thailand bamboo shoot shell fish (clouding Oxyeleotris marmoratus
Oxyeleotris marmoratus) and local bamboo shoot shell fish (rivers and creeks Odontobutis obscura
Odontobutis potamophila) fry differentiates.
Background technology
The clouding Oxyeleotris marmoratus (
Oxyeleotris marmoratus) popular name Thailand bamboo shoot shell fish (Marble goby), belong to Perciformes (Pereifomes), Yellowfin goby suborder (Gobioidei), Eleotridae (Eleotridae), Oxyeleotris marmoratus belong to (
Oxyeleotris), be build maximum in the Eleotridae fish, the fastest famous and precious fresh water fingerling of growth.The clouding Oxyeleotris marmoratus is to originate in countries in Southeast Asia such as Thailand, Vietnam, Malaysia, also is simultaneously one of improved seeds of these country's foreign exchange earnings.Because characteristics such as this fishing gear has that decorative pattern is beautiful, delicious flavour, mouthfeel are soft, and is nutritious are subjected to China coastal cities and Hong Kong, Macao, Taiwan human consumer's welcome deeply.China has now become the important freshwater aquiculture object in coastal areas of southern China area since last century, the eighties was introduced a fine variety from South East Asia.In recent years, along with the expansion of the scale of breed, the reduction of the cost of surviving the winter and the maturation of cultural technique, the breed of this fish is just risen on province, the middle and lower reach of Yangtze River and other places, and the trend that enlarges and northwards extend is year by year arranged.
The rivers and creeks Odontobutis obscura (
Odontobutis potamophila), be under the jurisdiction of Perciformes (Perciformes), Yellowfin goby suborder (Gobioidei), Eleotridae (Odontobutidae), Odontobutis (
Odontobutis), popular name sleeper fish also is called local bamboo shoot shell fish by the people on ground such as Jiangsu, is a kind of comparatively famous and precious small-sized economic fish.It mainly is distributed in the Changjiang river, downstream and riverine each tributary, Min River water system, Qiantang River water system, accidental the Yellow River water system.The rivers and creeks Odontobutis obscura is subjected to liking of ground people such as Jiangsu and Zhejiang Provinces, Shanghai, Fujian deeply because thorn between its fine and tender taste, delicious flavour, flesh is few.Simultaneously, this fish also is to have one of fish species of good development prospect in Jiangsu Province's characteristic water industry.
Because clouding Oxyeleotris marmoratus and rivers and creeks Odontobutis obscura differ from one another, and market demand grows with each passing day, so the breed scale of the two kinds of fishes in ground such as Jiangsu is also enlarging year by year, the seed demand also significantly increases thereupon, but the consequent is the source problem that comes of seed.Because clouding Oxyeleotris marmoratus and rivers and creeks Odontobutis obscura are very high in fry stage similarity, with the naked eye also be difficult to its resolution is come even culture veteran fisherman.So under the trend of economic interests, dragons and fishes jumbled together in domestic bamboo shoot shell fry kind market.The clouding Oxyeleotris marmoratus is that China is from country in Southeast Asia's introduced variety; though it is individual big; but tolerance to cold is far away from the rivers and creeks Odontobutis obscura; and water temperature be lower than 10 the degree field conditionss under be difficult to the survival, it is wild that the rivers and creeks Odontobutis obscura then mostly is, domestic then the needs tames it; and maximum individuality only is about 200g; at present, this fish is artificial propagation in scale success in the THE LOWER YANGTZE VALLEY province, and cultured area constantly enlarges.If the culturist gets kind wrong accidentally, certainly will cause post-processed improper, thus, the financial loss that the raiser is caused is with inestimable.Therefore, have a extensive future but under the background that fry market mixes in breed, press for and a kind of two kinds of bamboo shoot shell fish fries are differentiated the technology that comes, avoid culturing error and occur in the source.
Microsatellite DNA in the genome is with the heredity of Mendelian's mode, being codominance expresses, and have simultaneously distribute wide, genetic information content is high and be convenient to advantage such as PCR detections, has been widely used in the research of aspects such as the evaluation of fish sibship, analysis of genetic diversity, genetic linkage map structure and phyletic evolution.Studies show that little satellite flanking sequence between kind and have between the genus of nearer sibship quite conservatively, and has the part microsatellite locus between sibship taxonomical group far away certain conservative property to be arranged also in genome in belonging to.The nearly edge species that the conservative property of little satellite flanking sequence makes the micro-satellite primers developed in a certain species be applied to be correlated with become possibility.
Summary of the invention
The invention provides the discrimination method of a kind of Thailand bamboo shoot shell fish and local bamboo shoot shell fish fry.Fast two kinds of fries are distinguished by molecule means (1 pair of micro-satellite primers), avoid because seed is chosen and improperly brings financial loss to the raiser.
Ultimate principle of the present invention is: (1) develops clouding Oxyeleotris marmoratus micro-satellite primers in a large number with efficient enrichment with magnetic bead method; (2) from the exploitation primer, be chosen at the several amplifications that micro-satellite primers are used for rivers and creeks Odontobutis obscura colony that to stablize amplification in the clouding Oxyeleotris marmoratus colony and not have polymorphism; (3) filter out in rivers and creeks Odontobutis obscura colony and also can obviously increase, but the amplified band position obviously is different from the micro-satellite primers of clouding Oxyeleotris marmoratus, be used for the discriminating of clouding Oxyeleotris marmoratus and rivers and creeks Odontobutis obscura fry.
The present invention filters out the primer that a pair of kind is used for Thailand bamboo shoot shell fish and the discriminating of local bamboo shoot shell fish fry, called after H228, and primer sequence is as follows:
F：TCCAATCAGGCAGTAAGCAG（SEQ ID No.8）
；
R：GGGACATCAGGACCAACTCT（SEQ ID No.9）
。
Method with above-mentioned H228 primer discriminating Thailand bamboo shoot shell fish and local bamboo shoot shell fish fry comprises the following steps:
(1) with the H228 primer sample to be identified is increased, obtain the PCR product;
(2) with 1% agarose gel electrophoresis the PCR product is detected, EB dyeing, the 250bp size strip be Thailand bamboo shoot shell fish, the 300bp size strip be local bamboo shoot shell fish;
H228 primer of the present invention obtains according to following steps: 1. the extraction of genomic dna (clouding Oxyeleotris marmoratus colony, rivers and creeks Odontobutis obscura colony); 2. the enrichment with magnetic bead method is screened the little point of the little satellite of clouding Oxyeleotris marmoratus; 3. screening is the singlet site in clouding Oxyeleotris marmoratus colony; 4. expand rivers and creeks Odontobutis obscura colony with singlet site primer; 5. filter out 1 pair and all can stablize amplification two colonies, and the visibly different primer of clip size, be used for the discriminating of clouding Oxyeleotris marmoratus and rivers and creeks Odontobutis obscura fry.
Described step 1, the extraction of genomic dna comprises: tail fin 0.1 ~ 0.2g of clip experiment fish, take conventional phenol-chlorine method to extract genomic dna; The nucleic acid-protein determinator is measured its purity and concentration, and agarose gel electrophoresis detects the DNA integrity.
Described step 2, the little point of the enrichment with magnetic bead method screening little satellite of clouding Oxyeleotris marmoratus: with clouding Oxyeleotris marmoratus genomic dna warp
Bsp143The I restriction enzyme carries out enzyme and cuts the back jointing, carries out enrichment with magnetic bead behind the pcr amplification, and enriched product checks order to positive colony after cloning.
Described step 3, screening is the singlet site in clouding Oxyeleotris marmoratus colony: the sequence that contains the little point of little satellite in to sequencing result is carried out design of primers, pcr amplification is carried out in the synthetic back of primer in clouding Oxyeleotris marmoratus colony, amplified production screens through 6% polyacrylamide gel electrophoresis, select and only amplify a clear band, namely present the micro-satellite primers of singlet.
Described step 4, expand rivers and creeks Odontobutis obscura colony with singlet site primer: with the primer that screens Odontobutis obscura colony in rivers and creeks is carried out pcr amplification, in the amplification procedure, annealing temperature, MgCl
2Condition such as concentration and the amplification of clouding Oxyeleotris marmoratus colony are identical.
Described step 5, filter out two colonies and all can stablize amplification, and the visibly different primer of clip size, be used for the discriminating of clouding Oxyeleotris marmoratus and rivers and creeks Odontobutis obscura fry: detect clouding Oxyeleotris marmoratus colony and rivers and creeks Odontobutis obscura colony pcr amplification product simultaneously with 1% agarose gel electrophoresis, filter out the micro-satellite primers that to differentiate clouding Oxyeleotris marmoratus colony and rivers and creeks Odontobutis obscura colony.
51 ℃ of the micro-satellite primers H228(annealing temperatures that the present invention obtains), in clouding Oxyeleotris marmoratus group He in the rivers and creeks Odontobutis obscura colony, all can stablize amplification, the result is stable, band is clear, only need just can see notable difference with the detection of 1% agarose gel electrophoresis, and simple to operation, cost is low, practical strong, helpful to breeding production.
Description of drawings
Fig. 1: the partial results that H13 increases in clouding Oxyeleotris marmoratus colony (polyacrylamide gel electrophoresis screening singlet primer)
Fig. 2: the amplification (agarose gel electrophoresis screening diagnostic primers) of H228 microsatellite marker in part clouding Oxyeleotris marmoratus colony and rivers and creeks Odontobutis obscura colony.
Embodiment
1. the extraction of genomic dna (clouding Oxyeleotris marmoratus colony, rivers and creeks Odontobutis obscura colony):
1) gets the tail fin 0.1 ~ 0.3g that tests fish and add 450 μ L DNA extraction buffer (10 mmol/L Tris-HCl, 5 mmol/L EDTA, 75 mmol/L NaCl), add SDS and Proteinase K to final concentration behind the mixing successively and be respectively 0.5% and 200 μ g/mL, spend the night in 55 ℃ of water-bath digestion then.
2) add the saturated phenol of equal-volume, phenol/chloroform/primary isoamyl alcohol (25:24:1), each extracting of chloroform/primary isoamyl alcohol (24:1) once.After each extracting all softly mixed 10 minutes, 4 ℃, centrifugal 20 minutes of 12000rpm got supernatant.
3) the gained supernatant liquor is added the dehydrated alcohol precipitation of 2 times of volumes of 2.5 mol/L NaCl (final concentration is 0.2 mol/L) and precooling, 12000rpm got precipitation in centrifugal 20 minutes; With seasoning at room temperature after the 70% ethanol rinsing 2 times, treat to be dissolved among the TE of 200 μ L after the ethanol volatilization, place 4 ℃ of refrigerators standby.Measure its purity and concentration at the nucleic acid-protein determinator, agarose gel electrophoresis detects.
2. the enrichment with magnetic bead method is screened the little point of the little satellite of clouding Oxyeleotris marmoratus:
1) with clouding Oxyeleotris marmoratus genomic dna warp
Bsp143Four kinds of sticking end limit restriction endonucleases of I carry out the single endonuclease digestion reaction.Filter out the enzyme that endonuclease bamhi mainly is distributed in the 400-1200bp scope and cut system.Enzyme is cut system: genomic dna 8 μ g, 10 * buffer, 4 μ L, restriction endonuclease 5U, cumulative volume 40 μ L.Reaction conditions is: 37 ℃ of water bath with thermostatic control 5h, enzyme cut and finish 20 minutes termination reactions of back 65 ℃ of water-baths.Get 5 μ L enzymes and cut the detection of product sepharose.Use the AxyPrep dna gel recovery test kit recovery enzyme of Axygen company to cut product.
2) the DNA two ends of reclaiming are connected phosphorylated linker Linker A, joint sequence is:
Linker A 5’-GCGGTACCCGGGAAGCTTGG-3’（SEQ ID No.1）
3’-CGCCATGGGCCCTTCGAACCCTAG-p-5’ （SEQ ID No.2）
Joint long-chain sticking terminal (5 ' end) band phosphate radical.Get enzyme and cut back recovery DNA 14 μ L, add Linker A (100 μ M) 2 μ L, connect buffer 2 μ L, the T4 ligase enzyme, cumulative volume 20 μ L, 16 ℃ of connections are spent the night.Connect product and reclaim through AxyPrep DNA test kit, remove unnecessary joint, use 40 μ L elutriant wash-outs, get 5 μ L electrophoresis detection.
3) remaining elutriant is used for Pre-PCR, makes 10 pipe PCR altogether, adds 10 * Taq buffer, 2 μ L in each reaction tubes, dNTP(2mM) 1 μ L, MgCl
2(25mM) 1.5 μ L, primer Primer I(10 μ M) 1 μ L, Taq enzyme 1U, template DNA 3.5 μ L, cumulative volume are 20 μ L.The sequence of Primer I is: 5 '-GCGGTACCCGGGAAGCTTGG-3 ' (SEQ ID No.3).The PCR reaction is 12 circulations, and each circulation comprises: 94 ℃ of sex change 30s, and 63 ℃ of annealing 30s, 72 ℃ are extended 60s.The pre-PCR product agarose electrophoresis that takes a morsel after reaction finishes detects.Remain about 200 μ L reaction solutions and reclaim through AxyPrep DNA test kit, wash-out is in the elutriant of 40 μ L.
4) biotinylated probes and library hybridization, Streptomycin sulphate enrichment with magnetic bead and Post-PCR: get the DNA elutriant of whole 40 μ L, add ddH
2O 460 μ L place 95 ℃ of water-bath sex change 5 minutes with reaction tubes; Simultaneously with the biotinylated probes Biotin(CA of 50 μ M) 3 μ L add in another EP pipe, and add 20 * SSC(8.8g NaCl, 4.4g Trisodium Citrate, be configured to 100mL solution, regulate pH 7.2 back sterilizations) 13 μ L, 68 ℃ of preheatings add and finish the dna solution of denaturing step and continue 68 ℃ of insulation hybridization 10 minutes, place the room temperature cooling afterwards.The sequence of biotinylated probes Biotin (CA) is as follows:
Biotin-5’-ATAGAATATCACACACACACACACACACACACACACACACA-3’ （SEQ ID No.4）
(Promega) carries out the fragment enrichment with magnetic bead, needs washing before magnetic bead uses: flick the pipe end, the suspension magnetic bead utilizes magnet stand Separation Stand to collect, and abandons solution; Add 300 μ L, 0.5 * SSC in pipe, flick pipe end mixing, magnet stand is abandoned solution (washing 3 times) after collecting; It is stand-by to add 100 μ L, 0.5 * SSC and resuspended magnetic bead.In the magnetic bead pipe that washing finishes, add full dose and hybridize good dna solution, put upside down mixing once in every 1-2 minute, mixing 5-10 time; Utilize magnet stand to collect magnetic bead, sucking-off solution is retained (prevent that the magnetic bead inefficacy from causing the failure of an experiment, retain solution and magnetic bead reaction newly as available this of Post-PCR reaction failure), uses 300 μ L0.1 * SSC to wash magnetic bead, wash 4 times, exhaust solution for the last time as far as possible; Add 100 μ L ddH
2O, resuspended magnetic bead, 95 ℃ of water-baths 5 minutes utilize magnet stand to collect magnetic bead, change dna solution over to new pipe; Utilize vacuum-drying instrument or baking oven that 100 μ LDNA liquid are concentrated into about 20 μ L.
Post-PCR makes 5 pipes altogether, adds 10 * Taq buffer, 2 μ L in each reaction tubes, dNTP(2mM) 1 μ L, MgCl
2(25mM) 1.5 μ L, primer Primer I(10 μ M) 1 μ L, Taq enzyme 1U, template DNA 4 μ L, cumulative volume are 20 μ L.The PCR reaction is 12 circulations, and each circulation comprises: 94 ℃ of sex change 30s, and 63 ℃ of annealing 30s, 72 ℃ are extended 60s.The agarose electrophoresis that takes a morsel the PCR reaction solution detects, and all the other reclaim through AxyPrep DNA test kit, and wash-out is in the elutriant of 40 μ L and use the nucleic acid-protein instrument to measure DNA concentration.
3. screening is the singlet site in clouding Oxyeleotris marmoratus colony:
1) according to the concentration of gained DNA, the ratio of regulating foreign DNA and T carrier: carrier DNA is 1:2-10 with the ratio scope of inserting the fragment mole number.Linked system is: foreign DNA 1-4 μ L, and T carrier (0.03pmol) 1 μ L connects buffer 5 μ L, cumulative volume 10 μ L, 16 ℃ of connections are spent the night.
2) full dose 10 μ L are connected liquid and add in the 100 μ L DH5a competent cells, placed on ice 30 minutes; Placed on ice immediately 1 minute behind 42 ℃ of heat shock 60s; Add 900 μ L LB substratum, 37 ℃ of 100rpm concussions were cultivated 60 minutes; Get 100 μ L bacterium liquid evenly be layered on contain X-gal (40 μ L, 20mg/mL), IPTG(7 μ L, 200mg/mL), Amp(final concentration 60 μ g/mL) the 90mL agar plate on, treat that liquid is absorbed fully by agar plate after, cultivated 12-15 hour for 37 ℃; The single white colony of picking is inoculated in 500 μ L and contains in the LB liquid nutrient medium of 60 μ g/mL Amp 220rpm shaking culture 3 hours.
3) use M13 and RV-M primer to be bacterium liquid PCR, whether the bacterial detection clone contains the insertion fragment, and reaction conditions is 10 * Taq buffer, 1 μ L, dNTP(2mM) 0.5 μ L, MgCl
2(25mM) 0.8 μ L, primer+(10 μ M) 0.3 μ L, primer-(10 μ M) 0.3 μ L, Taq enzyme 0.5U, bacterium liquid 0.5 μ L, cumulative volume are 10 μ L.The PCR reaction is 25 circulations, and each circulation comprises: 94 ℃ of sex change 30s, and 56 ℃ of annealing 30s, 72 ℃ are extended 60s.Agarose gel electrophoresis detects, and chooses the positive colony of amplified fragments 400-1200bp scope and does the secondary PCR screening.
M13： 5’-CGCCAGGGTTTTCCCAGTCACGAC-3’ （SEQ ID No.5）
RV-M：5’-GAGCGGATAACAATTTCACACAGG-3’ （SEQ ID No.6）
4) postsearch screening adopt in M13, the RV-M primer one with the PrimerII-CA primer that partners, two combination: M13+PrimerII-CA altogether, RV-M+PrimerII-CA.Reaction conditions is 10 * Taq buffer, 1 μ L, dNTP(2mM) 0.5 μ L, MgCl
2(25mM) 0.8 μ L, primer+(10 μ M) 0.3 μ L, primer-(10 μ M) 0.3 μ L, Taq enzyme 0.5U, bacterium liquid 0.5 μ L, cumulative volume are 10 μ L.The PCR reaction is 25 circulations, and each circulation comprises: 94 ℃ of sex change 30s, and 56 ℃ of annealing 30s, 72 ℃ are extended 60s.Agarose gel electrophoresis detects, and chooses the corresponding bacterium liquid that amplified fragments occurs and send biotech firm's order-checking.
PrimerII-CA：5’-CACACACACACACACACACACACA-3’ （SEQ ID No.7）
5) contain the sequences Design primer of microsatellite locus with 5.0 couples of Primer, primer is synthetic by Shanghai Jierui Biology Engineering Co., Ltd.The synthetic back of primer is used for clouding Oxyeleotris marmoratus colony pcr amplification.System is: 10 * Taq buffer, 2 μ L, dNTP(2mM) 2 μ L, MgCl
2(25mM) 1.5 μ L, primer+(10 μ M) 1 μ L, primer-(10 μ M) 1 μ L, Taq enzyme 1U, template DNA 1 μ L adds the sterilization distilled water and supplies 20 μ L systems.The PCR program is 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 30sec, and annealing 30sec, 72 ℃ are extended 30sec, react 30 circulations, and last 7 ℃ are extended 5min.
6) the PCR amplified production is through 8% polyacrylamide gel electrophoresis, power 80W, and electrophoresis 1.5h, cma staining, camera is taken pictures, filters out in clouding Oxyeleotris marmoratus colony, to be the many to micro-satellite primers of singlet, be example with H13, effect is as shown in Figure 1.
4. expand rivers and creeks Odontobutis obscura colony with singlet site primer:
The micro-satellite primers that screens with previous step carries out pcr amplification to rivers and creeks Odontobutis obscura colony, reaction conditions and system and identical in clouding Oxyeleotris marmoratus colony, and namely system is: 10 * Taq buffer, 2 μ L, dNTP(2mM) 2 μ L, MgCl
2(25mM) 1.5 μ L, primer+(10 μ M) 1 μ L, primer-(10 μ M) 1 μ L, Taq enzyme 1U, template DNA 1 μ L adds the sterilization distilled water and supplies 20 μ L systems.The PCR program is 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 30sec, and annealing 30sec, 72 ℃ are extended 30sec, react 30 circulations, and last 7 ℃ are extended 5min.Though little satellite flanking sequence has suitable conservative property, because not belonging to, rivers and creeks Odontobutis obscura and clouding Oxyeleotris marmoratus do not belong to fish together, still have the part primer in rivers and creeks Odontobutis obscura colony, to increase.
5. filter out 1 pair and all can stablize amplification two colonies, and the visibly different primer of clip size, be used for the discriminating of clouding Oxyeleotris marmoratus and rivers and creeks Odontobutis obscura fry: will all have stable amplification PCR products to detect with 1% agarose gel electrophoresis two colonies, EB dyeing, it is clear to filter out band, and the position is obviously different, can be used in a pair of micro-satellite primers of the discriminating of clouding Oxyeleotris marmoratus and rivers and creeks Odontobutis obscura fry, called after H228, primer sequence is shown in SEQ ID No.8 and SEQ ID No.9.The amplification of H228 microsatellite marker in clouding Oxyeleotris marmoratus colony and rivers and creeks Odontobutis obscura colony as
Fig. 2,The 250bp size be Thailand bamboo shoot shell fish, the 300bp size be local bamboo shoot shell fish.
SEQUENCE LISTING
＜110〉Nanjing Normal University
＜120〉diagnostic primers and the method for a kind of Thailand bamboo shoot shell fish and local bamboo shoot shell fish fry
<130>
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
＜213〉artificial sequence
<400> 1
<210> 2
<211> 24
<212> DNA
＜213〉artificial sequence
<400> 2
cgccatgggc ccttcgaacc ctag 24
<210> 3
<211> 20
<212> DNA
＜213〉artificial sequence
<400> 3
<210> 4
<211> 41
<212> DNA
＜213〉artificial sequence
<400> 4
atagaatatc acacacacac acacacacac acacacacac a 41
<210> 5
<211> 24
<212> DNA
＜213〉artificial sequence
<400> 5
cgccagggtt ttcccagtca cgac 24
<210> 6
<211> 24
<212> DNA
＜213〉artificial sequence
<400> 6
gagcggataa caatttcaca cagg 24
<210> 7
<211> 24
<212> DNA
＜213〉artificial sequence
<400> 7
cacacacaca cacacacaca caca 24
<210> 8
<211> 20
<212> DNA
＜213〉artificial sequence
<400> 8
<210> 9
<211> 20
<212> DNA
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<400> 9
Claims (2)
1. one kind is used for the primer that clouding Oxyeleotris marmoratus and rivers and creeks Odontobutis obscura fry are differentiated, it is characterized in that described primer sequence is as follows:
F：TCCAATCAGGCAGTAAGCAG；
R：GGGACATCAGGACCAACTCT。
2. the discrimination method of a clouding Oxyeleotris marmoratus and rivers and creeks Odontobutis obscura fry is characterized in that, comprises the following steps:
(1) with the described primer of claim 1 sample to be identified is increased, obtain the PCR product;
(2) with 1% agarose gel electrophoresis the PCR product is detected, EB dyeing, the 250bp size strip be the clouding Oxyeleotris marmoratus, the 300bp size strip be the rivers and creeks Odontobutis obscura.
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| CN103667499B (en) * | 2013-12-26 | 2015-01-28 | 南京师范大学 | Primer and method for identifying odontobutis potamophila, o. yaluensis and o. sinensis |
| CN104531847A (en) * | 2014-12-10 | 2015-04-22 | 南京师范大学 | Microsatellite marker method for identifying wild odontobutis potamophila population of minjiang river basin |
| CN112391481B (en) * | 2020-11-18 | 2021-08-17 | 中国水产科学研究院珠江水产研究所 | SNPs for identifying fish species of oxyeleotris obscura, AS-PCR primer group based on SNPs, and detection method and application thereof |
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