CN111321231B - Method for molecular identification of rainbow trout gynogenesis offspring - Google Patents
Method for molecular identification of rainbow trout gynogenesis offspring Download PDFInfo
- Publication number
- CN111321231B CN111321231B CN202010177529.6A CN202010177529A CN111321231B CN 111321231 B CN111321231 B CN 111321231B CN 202010177529 A CN202010177529 A CN 202010177529A CN 111321231 B CN111321231 B CN 111321231B
- Authority
- CN
- China
- Prior art keywords
- offspring
- rainbow trout
- gel
- gynogenesis
- electrophoresis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a method for molecular identification of a rainbow trout gynogenesis offspring, which comprises the following steps: and extracting genome DNA from tail fins of the female nucleus development offspring of the American red salmon and the rainbow trout, and identifying female nucleus development offspring of the rainbow trout through a PCR reaction system and a program, PCR product agarose gel electrophoresis detection, polyacrylamide gel electrophoresis and silver staining and mark verification of whether a lane on a display strip contains male parents. According to the invention, the screening of the gynogenesis offspring of the rainbow trout is carried out by applying the screened molecular marker AOMM108, the gynogenesis offspring can be identified earlier from the juvenile rainbow trout stage, the economic loss caused by the later culture period is reduced, and the breeding effect is improved. The method is simple and quick, has high accuracy, and can play an important role in the field of rainbow trout genetic breeding.
Description
Technical Field
The invention belongs to the technical field of aquatic genetic breeding, and particularly relates to a method for molecular identification of a rainbow trout gynogenesis offspring.
Background
The fish gynogenesis technology has extremely important application value in the field of aquaculture, namely, after the ovum is activated by genetically inactivated sperm, the gynogenesis of the fish is artificially induced, and then the gynogenesis of the fish is developed into a special sexual reproduction mode of the filial generation by inhibiting polar body exclusion or first cleavage of the fertilized ovum. The male fish of rainbow trout is generally sexually mature at 2 years old and the female fish is sexually mature at 3 years old. The mature individuals show the characteristics of reduced growth rate, increased death rate, poorer meat quality and appearance and the like, and are unfavorable for cultivation production. The offspring obtained by adopting the sperms of the American red salmon to carry out heterogeneous sperm induction rainbow trout gynogenesis is the population of female fish, so that adverse effects can be reduced, and meanwhile, a breeding material can be provided for the next breeding work. However, the external morphology of the experimental offspring is difficult to distinguish whether the experimental offspring are truly gynogenesis offspring, especially in juvenile fish stage, and no effective means is available at present for accurately identifying the gynogenesis offspring of rainbow trout. Since individuals of the filial generation are sterile, great waste is caused to the breeding work, which is a difficult problem in the rainbow trout breeding work, and therefore, the early efficient and accurate identification of the rainbow trout gynogenesis offspring is a core problem to be solved.
Disclosure of Invention
The invention aims at solving the technical problems in the prior art and provides a method for molecular identification of the gynogenesis offspring of rainbow trout.
The technical scheme adopted for solving the technical problems of the invention is as follows:
a method for molecular identification of the gynogenesis offspring of rainbow trout comprises the following steps: extracting genome DNA from tail fins of the female nucleus development offspring of the American red salmon and the rainbow trout, and identifying female nucleus development offspring conditions of the rainbow trout through a PCR reaction system and a program, PCR product agarose gel electrophoresis detection, polyacrylamide gel electrophoresis and silver staining and mark verification of whether a lane on a display strip contains male parents;
wherein the primer in the PCR reaction system is AOMM108,
F:GGAGGCAGCTCCTGTTTTC，R:GCTGCGCCATCTCTCTATCT。
the PCR reaction system is as follows: the total system was 13. Mu.L, of which 2-fold concentration was 6.5. Mu.L of PCR premix enzyme;TaqDNA polymerase: 0.1U/ML; magnesium chloride: 4mmol/L; dNTP mixture: 0.4mmol/L, two primers of 10mmol/L each 0.5. Mu.L; 0.5 Mu L of DNA template; the rest double distilled water is complemented;
the PCR reaction procedure was: pre-denaturation at 94℃for 3 min; pre-denaturation at 94℃for 35 s, annealing at 59℃for 45 s, extension at 72℃for 45 s for 30 cycles; after the reaction was completed, the reaction was extended at 72℃for 8 minutes.
The method for identifying the rainbow trout gynogenesis offspring by the molecules comprises the following specific steps:
(1) Extraction and detection of DNA
Collecting tail fin samples of the female gynogenesis offspring of the American red salmon and the rainbow trout, and extracting genome DNA by adopting a high salt concentration extraction method;
(2) Primer design
The 1 pair of primers are AOMM108, F GGAGGCAGCTCCTGTTTTCT and R GCTGCGCCATCTCTCTATCT;
(3) PCR reaction system and program
Amplifying the target fragment by using a synthesized upstream and downstream primer, wherein the PCR reaction system is as follows: the total system was 13. Mu.L, including 13. Mu.L, of which 2-fold concentration was 6.5. Mu.L of PCR premix enzyme; taqDNA polymerase: 0.1U/ML; magnesium chloride: 4mmol/L; dNTP mixture: 0.4mmol/L, two primers of 10mmol/L each 0.5. Mu.L; 0.5 Mu L of DNA template; the rest double distilled water is complemented;
the PCR reaction procedure was: pre-denaturation at 94℃for 3 min; pre-denaturation at 94℃for 35 s, annealing at 59℃for 45 s, extension at 72℃for 45 s for 30 cycles; after the reaction is finished, the mixture is further extended for 8 minutes at the temperature of 72 ℃;
(4) Agarose gel electrophoresis detection of PCR products
Electrophoresis of the amplified product in agarose gel with mass fraction of 10% and 1 xTAE buffer at 5V/cm voltage for 15min, and detecting whether there is a band;
(5) Polyacrylamide gel electrophoresis and silver staining
Preparing gel by using double distilled water, acrylamide, 5 XTBE, tetramethyl ethylenediamine TEMED and AP, injecting the prepared gel into a gel tank, inserting a comb, standing for solidification, removing the comb, assembling the comb on an electrophoresis tank, loading a sample of a product, taking out the gel after electrophoresis, putting the gel into a porcelain tray filled with pure water, washing once, adding silver nitrate, placing the porcelain tray on a shaking table for dyeing, pouring out the silver nitrate, adding pure water for washing, adding NaOH solution into the magnetic tray, placing the magnetic tray for dyeing, starting to present a strip, pouring the NaOH solution, adding pure water for washing, spreading a film on a film observing box, and photographing and recording;
(6) Mark verification
The results show that the lanes of the gynogenesis offspring do not contain the bands of the male parent, the genetic material which can be identified as the gynogenesis offspring comes from the female parent, and the genetic material of the male parent is not found to infiltrate; the results show that bands containing male parents in lanes of the gynogenesis offspring can be identified as offspring.
The primer design in the step (2) is as follows: according to the sequence information with accession number AF352758.1 in GenBank database, 1 pair of primers AOMM108, F: GGAGGCAGCTCCTGTTTTCT, R: GCTGCGCCATCTCTCTATCT, which were synthesized by Shanghai Bioengineering technologies, inc. were designed with Primer3, v.0.4.0.
The electrophoresis conditions in the step (5) are as follows: the electrophoresis buffer solution is 1 XTBE, the voltage is 200V, and the electrophoresis time is 2.5-3h.
The specific method for extracting and detecting the DNA in the step (1) comprises the following steps:
a. digestion
Taking 0.5-1g of tail fin samples of the American red salmon and rainbow trout gynogenesis offspring, adding the tail fin samples into a centrifuge tube, adding 500ul of Hom Buffer and 15ul of proteinase K, putting the mixture into a water bath kettle at 55-60 ℃ for 2-3h, and shaking uniformly every 30 minutes;
b. extraction
Adding 500ul of NaCL and 300ul of chloroform with the concentration of 4.5mol/L into a centrifuge tube, continuously shaking for 15min, centrifuging for 10min, and sucking 800ul of supernatant into a new centrifuge tube after centrifuging;
c. precipitation and drying
Adding 595ul of isopropanol into a new centrifuge tube, uniformly mixing for 1-2 minutes, standing at-20 ℃ for precipitation for 2 hours, centrifuging at 12000-13000rpm for 10 minutes, removing supernatant, adding 500ul of ethanol with the volume fraction of 70% for washing for 5 minutes, centrifuging for 10 minutes, removing supernatant, placing in a sterile super clean bench for naturally airing, adding 60ul of sterile double distilled water into the aired centrifuge tube, fully dissolving DNA, and preserving at-20 ℃ for standby;
the centrifugation speed in the step b and the step c is 12000-13000rpm/min.
The specific method for the polyacrylamide gel electrophoresis and silver staining in the step (5) is as follows: taking 25.6ml of double distilled water, 18.4ml of acrylamide with the mass fraction of 30%, 11ml of 5 XTBE, 0.134ml of tetramethyl ethylenediamine TEMED with the mass fraction of 10% of AP (polyethylene glycol) 350ul, uniformly stirring to prepare gel, injecting the prepared gel into an assembled gel tank, inserting a comb, standing for 2-3h for solidification, taking the comb out, assembling the comb onto an electrophoresis tank, taking out the gel after the PCR product is loaded into 5 mu L, putting the gel into a porcelain dish filled with pure water after electrophoresis is completed, adding 800ml of silver nitrate with the mass fraction of 1% after washing once, placing the silver nitrate on a shaking table for dyeing for 8-10 min, pouring out the silver nitrate, adding pure water for rapid washing for 10s, adding 1000ml of NaOH solution with the mass fraction of 2% into the magnetic disk, placing the gel on the shaking table for dyeing for 10min, starting to present a strip, pouring the NaOH solution, adding the pure water for washing, spreading the strip on a film viewing box, and photographing and recording.
The beneficial effects of the invention are as follows: the molecular marker AOMM108 is used for screening the gynogenesis offspring of the rainbow trout, the gynogenesis offspring can be identified earlier from the juvenile stage of the rainbow trout, the economic loss caused by the later stage of cultivation is reduced, and the breeding effect is improved. The method is simple and quick, has high accuracy, and can play an important role in the field of rainbow trout genetic breeding.
Drawings
FIG. 1 is an amplification map of the gynogenesis offspring and its parents in the microsatellite molecular marker AF352758.1 used according to the invention; gynogenesis individuals: lanes 1-13, M: marker, male parent: M14-M17, female parent: F18-F21.
Detailed Description
The invention will be described in detail below with reference to the drawings and the detailed description.
Example 1
A method for molecular identification of the gynogenesis offspring of rainbow trout comprises the following specific steps:
1. extraction and detection of DNA
Collecting a sample, taking a tail fin sample of the American red salmon and rainbow trout gynogenesis offspring 0.5-g, and extracting genome DNA by adopting a high salt concentration extraction method.
1.1 Digestion
Adding tail fin tissue into a centrifuge tube, adding 500ul of Hom Buffer and 15ul of proteinase K, placing into a water bath kettle at 60 ℃ for 3 hours, and shaking uniformly every 30 minutes;
1.2 Extraction
Adding 500ul of NaCL and 300ul of chloroform of 4.5mol/L into a centrifuge tube, shaking uniformly for 15min, centrifuging for 10min at 12000rpm/min, and sucking 800ul of supernatant into a new centrifuge tube after centrifuging;
1.3 Precipitation and drying
Adding 595ul of isopropanol, mixing for 1 min, standing at-20deg.C for precipitation for 2 hr, centrifuging at 13000rpm/min for 10min, removing supernatant, adding 500ul of 70% ethanol, washing for 5min, centrifuging at 12000rpm/min for 10min, removing supernatant, standing in sterile super clean bench, naturally airing, adding 60ul of sterile double distilled water in the aired centrifuge tube, dissolving DNA completely, and preserving at-20deg.C for use.
2. Primer design
Based on the sequence information with accession number AF352758.1 in GenBank database, 1 pair of primers AOMM108 (F: GGAGGCAGCTCCTGTTTTCT, R: GCTGCGCCATCTCTCTATCT) was designed with Primer3 (v.0.4.0) and synthesized by Shanghai Bioengineering services Co.
3. PCR reaction system and program
The target fragment was amplified using the synthesized upstream and downstream primers. PCR reaction system: the total system was 13. Mu.L, which included 6.5. Mu.L of dye-containing 2X Taq PCR Mastermix (TaqDNA Polymer: 0.1U/ML; mgCl) 2 :4mmol/L; dNTPSeach:0.4 mmol/L), two primers (10 mmol/L) 0.5. Mu.L, 0.5. Mu.L DNA template, and the remaining double distilled water were made up. The PCR reaction procedure was: pre-denaturation at 94℃for 3 min; pre-denaturation at 94℃for 35 s, annealing at 59℃for 45 s, extension at 72℃for 45 s for 30 cycles; after the reaction was completed, the reaction was extended at 72℃for 8 minutes.
4. Agarose gel electrophoresis detection of PCR product
The amplified product was electrophoresed in 10% by mass agarose gel and 1 XTAE buffer at 5V/cm for 15min, and the presence or absence of a band was detected.
5. Polyacrylamide gel electrophoresis and silver staining
Taking 25.6ml of double distilled water, 18.4ml of acrylamide with the mass fraction of 30%, 11ml of AP with the mass fraction of 5 multiplied by TBE,0.134ml TEMED,350ul, fully and uniformly stirring by using a glass rod, injecting prepared gel into an assembled gel tank, inserting a comb, standing for 3h for solidification, taking the comb off, and assembling the comb on an electrophoresis tank. The PCR product loading was 5. Mu.L. Electrophoresis conditions: the running buffer was 1 XTBE, voltage 200V, and running time 3h. After electrophoresis, the gel is taken out and put into a porcelain dish filled with pure water, and the gel is quickly cleaned once. Adding 800ml of silver nitrate with the mass fraction of 1%, placing on a shaking table for dyeing for 8 minutes, pouring out the silver nitrate, adding pure water, rapidly washing for 10 seconds, adding 1000ml of NaOH solution with the mass fraction of 2% into a magnetic disk, placing on the shaking table for dyeing for 10 minutes, starting to present a strip, pouring out the NaOH solution, adding the pure water for washing, spreading a film on a film viewing box, and photographing and recording.
6. Mark verification
The results show that the lanes of the gynogenesis offspring do not contain the bands of the male parent, the genetic material which can be identified as the gynogenesis offspring comes from the female parent, and the genetic material of the male parent is not found to infiltrate; the results show that bands containing male parents in lanes of the gynogenesis offspring can be identified as offspring.
Example 2
A method for molecular identification of the gynogenesis offspring of rainbow trout comprises the following specific steps:
1. extraction and detection of DNA
Collecting a sample, taking 1g of tail fin sample of the American red salmon and rainbow trout gynogenesis offspring, and extracting genome DNA by adopting a high salt concentration extraction method.
1.1 Digestion
Adding tail fin tissue into a centrifuge tube, adding 500ul of Hom Buffer and 15ul of proteinase K, placing into a water bath kettle at 55 ℃ for 2 hours, and shaking uniformly every 30 minutes;
1.2 Extraction
Adding 500ul of NaCL and 300ul of chloroform of 4.5mol/L into a centrifuge tube, shaking uniformly for 15min, centrifuging for 10min at the rotating speed of 13000rpm/min, and sucking 800ul of supernatant into a new centrifuge tube after centrifuging;
1.3 Precipitation and drying
Adding 595ul of isopropanol, mixing for 2 min, standing at-20deg.C for precipitation for 2 hr, centrifuging at 12000rpm/min for 10min, removing supernatant, adding 500ul of 70% ethanol, washing for 5min, centrifuging at 13000rpm/min for 10min, removing supernatant, naturally air drying in a sterile super clean bench, adding 60ul of sterile double distilled water in an air-dried centrifuge tube, dissolving DNA completely, and storing at-20deg.C for use.
2. Primer design
Based on the sequence information with accession number AF352758.1 in GenBank database, 1 pair of primers AOMM108 (F: GGAGGCAGCTCCTGTTTTCT, R: GCTGCGCCATCTCTCTATCT) was designed with Primer3 (v.0.4.0) and synthesized by Shanghai Bioengineering services Co.
3. PCR reaction system and program
The target fragment was amplified using the synthesized upstream and downstream primers. PCR reaction system: the total system was 13. Mu.L, which included 6.5. Mu.L of dye-containing 2X Taq PCR Mastermix (TaqDNA Polymer: 0.1U/ML; mgCl) 2 :4mmol/L; dNTPasach 0.4 mmol/L), two primers (10 mmol/L) 0.5 μL,0.5 μL DNA template, and the rest double distilled water. The PCR reaction procedure was: pre-denaturation at 94℃for 3 min; pre-denaturation at 94℃for 35 s, annealing at 59℃for 45 s, extension at 72℃for 45 s for 30 cycles; after the reaction was completed, the reaction was extended at 72℃for 8 minutes.
4. Agarose gel electrophoresis detection of PCR product
The amplified product was electrophoresed in 10% by mass agarose gel and 1 XTAE buffer at 5V/cm for 15min, and the presence or absence of a band was detected.
5. Polyacrylamide gel electrophoresis and silver staining
Taking 25.6ml of double distilled water, 18.4ml of acrylamide with the mass fraction of 30%, 11ml of AP with the mass fraction of 5 multiplied by TBE,0.134ml TEMED,350ul, fully and uniformly stirring by using a glass rod, injecting prepared gel into an assembled gel tank, inserting a comb, standing for 2h for solidification, taking the comb off, and assembling the comb on an electrophoresis tank. The PCR product loading was 5. Mu.L. Electrophoresis conditions: the running buffer was 1 XTBE, voltage 200V, and running time 2.5h. After electrophoresis, the gel is taken out and put into a porcelain dish filled with pure water, and the gel is quickly cleaned once. Adding 800ml of silver nitrate with the mass fraction of 1%, placing on a shaking table for dyeing for 10 minutes, pouring out the silver nitrate, adding pure water, rapidly washing for 10 seconds, adding 1000ml of NaOH solution with the mass fraction of 2% into a magnetic disk, placing on the shaking table for dyeing for 10 minutes, starting to present a strip, pouring out the NaOH solution, adding the pure water for washing, spreading a film on a film viewing box, and photographing and recording.
6. Mark verification
The results show that the lanes of the gynogenesis offspring do not contain the bands of the male parent, the genetic material which can be identified as the gynogenesis offspring comes from the female parent, and the genetic material of the male parent is not found to infiltrate; the results show that bands containing male parents in lanes of the gynogenesis offspring can be identified as offspring.
Claims (8)
1. A method for molecular identification of the gynogenesis offspring of rainbow trout is characterized in that the method comprises the following steps: extracting genome DNA from tail fins of the female nucleus development offspring of the American red salmon and the rainbow trout, and identifying female nucleus development offspring conditions of the rainbow trout through a PCR reaction system and a program, PCR product agarose gel electrophoresis detection, polyacrylamide gel electrophoresis and silver staining and mark verification of whether a lane on a display strip contains male parents; wherein the primer in the PCR reaction system is AOMM108, F: GGAGGCAGCTCCTGTTTTC, R: GCTGCGCCATCTCTCTATCT.
2. A method of molecular identification of the gynogenesis offspring of rainbow trout according to claim 1, characterized in that: the PCR reaction system is as follows: the total system was 13. Mu.L, of which 2-fold concentration was 6.5. Mu.L of PCR premix enzyme; taqDNA polymerase: 0.1U/ML; magnesium chloride: 4mmol/L; dNTP mixture: 0.4mmol/L, two primers of 10mmol/L each 0.5. Mu.L; 0.5 Mu L of DNA template; the rest double distilled water is complemented; the PCR reaction procedure was: pre-denaturation at 94℃for 3 min; pre-denaturation at 94℃for 35 s, annealing at 59℃for 45 s, extension at 72℃for 45 s for 30 cycles; after the reaction was completed, the reaction was extended at 72℃for 8 minutes.
3. A method for molecular identification of the gynogenesis offspring of rainbow trout according to claim 1 or 2, characterized in that the specific method is as follows: (1) Extracting DNA and detecting and collecting tail fin samples of the American red salmon and rainbow trout gynogenesis offspring, and extracting genome DNA by adopting a high salt concentration extraction method; (2) The primer design 1 is AOMM108, F is GGAGGCAGCTCCTGTTTTCT, R is GCTGCGCCATCTCTCTATCT; (3) And amplifying the target fragment by using a PCR reaction system and a program, wherein the PCR reaction system comprises the following steps: the total system is 13 mu L, wherein the concentration of the PCR premix enzyme is 2 times of that of the PCR premix enzyme is 6.5 mu L; taqDNA polymerase: 0.1U/ML; magnesium chloride: 4mmol/L; dNTP mixture: 0.4mmol/L, two primers of 10mmol/L each 0.5. Mu.L; 0.5 Mu L of DNA template; the rest double distilled water is complemented; the PCR reaction procedure was: pre-denaturation at 94℃for 3 min; pre-denaturation at 94℃for 35 s, annealing at 59℃for 45 s, extension at 72℃for 45 s for 30 cycles; after the reaction is finished, the mixture is further extended for 8 minutes at the temperature of 72 ℃; (4) Detecting the amplified product by agarose gel electrophoresis of the PCR product, and detecting whether a band exists or not in agarose gel with the mass fraction of 10% and 1 xTAE buffer solution by electrophoresis at a voltage of 5V/cm for 15 min; (5) Preparing gel by using double distilled water, acrylamide, 5 XTBE, tetramethyl ethylenediamine TEMED and AP, injecting the prepared gel into a gel tank, inserting a comb, standing for solidification, taking down the comb, assembling the comb on an electrophoresis tank, loading the product, taking out the gel after electrophoresis, putting the gel into a porcelain tray filled with pure water, washing once, adding silver nitrate, placing on a shaking table for dyeing, pouring out the silver nitrate, adding pure water for washing, adding NaOH solution into the magnetic disk, placing on the shaking table for dyeing, starting to present a strip, pouring out the NaOH solution, adding pure water for washing, spreading a film on a film viewing box, and photographing and recording; (6) The marking verification result shows that the lanes of the gynogenesis offspring do not contain the bands of the male parent, the genetic material which can be identified as the gynogenesis offspring comes from the female parent, and the genetic material of the male parent is not found to infiltrate; the results show that bands containing male parents in lanes of the gynogenesis offspring can be identified as offspring.
4. A method of molecular identification of the gynogenesis offspring of rainbow trout according to claim 3, characterized in that: the primer design in the step (2) is as follows: according to the sequence information with accession number AF352758.1 in GenBank database, 1 pair of primers AOMM108, F: GGAGGCAGCTCCTGTTTTCT, R: GCTGCGCCATCTCTCTATCT, which were synthesized by Shanghai Bioengineering technologies, inc. were designed with Primer3, v.0.4.0.
5. The method for molecular identification of the gynogenesis offspring of rainbow trout according to claim 4, wherein: the electrophoresis conditions in the step (5) are as follows: the electrophoresis buffer solution is 1 XTBE, the voltage is 200V, and the electrophoresis time is 2.5-3h.
6. The method for molecular identification of the gynogenesis offspring of rainbow trout according to claim 5, characterized in that the specific method for extracting and detecting the DNA of step (1) is as follows: a. digesting 0.5-1g of tail fin samples of the American red salmon and rainbow trout gynogenesis offspring, adding into a centrifuge tube, adding 500ul of Hom Buffer and 15ul of proteinase K, putting into a water bath kettle at 55-60 ℃ for 2-3h, and shaking uniformly every 30 minutes; b. extracting, namely adding 500ul of NaCL (sodium chloride) with the concentration of 4.5mol/L and 300ul of chloroform into a centrifuge tube, shaking uniformly for 15min, centrifuging for 10min, and sucking 800ul of supernatant into a new centrifuge tube after centrifuging; c. adding 595ul of isopropanol into a new centrifuge tube, uniformly mixing for 1-2 minutes, standing at-20 ℃ for precipitation for 2 hours, centrifuging at 12000-13000rpm for 10 minutes, removing supernatant, adding 500ul of ethanol with the volume fraction of 70% for washing for 5 minutes, centrifuging for 10 minutes, removing supernatant, placing in a sterile super clean bench for naturally airing, adding 60ul of sterile double distilled water into the aired centrifuge tube, fully dissolving DNA, and preserving at-20 ℃ for standby.
7. A method of molecular identification of the gynogenesis offspring of rainbow trout according to claim 6, wherein: the centrifugation speed in the step b and the step c is 12000-13000rpm/min.
8. A method of molecular identification of the gynogenesis offspring of rainbow trout according to claim 7, wherein: the specific method for the polyacrylamide gel electrophoresis and silver staining in the step (5) is as follows: taking 25.6ml of double distilled water, 18.4ml of acrylamide with the mass fraction of 30%, 11ml of 5 XTBE, 0.134ml of tetramethyl ethylenediamine TEMED with the mass fraction of 10% of AP (polyethylene glycol) 350ul, uniformly stirring to prepare gel, injecting the prepared gel into an assembled gel tank, inserting a comb, standing for 2-3h for solidification, taking the comb out, assembling the comb onto an electrophoresis tank, taking out the gel after the PCR product is loaded into 5 mu L, putting the gel into a porcelain dish filled with pure water after electrophoresis is completed, adding 800ml of silver nitrate with the mass fraction of 1% after washing once, placing the silver nitrate on a shaking table for dyeing for 8-10 min, pouring out the silver nitrate, adding pure water for rapid washing for 10s, adding 1000ml of NaOH solution with the mass fraction of 2% into the magnetic disk, placing the gel on the shaking table for dyeing for 10min, starting to present a strip, pouring the NaOH solution, adding the pure water for washing, spreading the strip on a film viewing box, and photographing and recording.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010177529.6A CN111321231B (en) | 2020-03-14 | 2020-03-14 | Method for molecular identification of rainbow trout gynogenesis offspring |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010177529.6A CN111321231B (en) | 2020-03-14 | 2020-03-14 | Method for molecular identification of rainbow trout gynogenesis offspring |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111321231A CN111321231A (en) | 2020-06-23 |
| CN111321231B true CN111321231B (en) | 2023-07-21 |
Family
ID=71169510
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010177529.6A Active CN111321231B (en) | 2020-03-14 | 2020-03-14 | Method for molecular identification of rainbow trout gynogenesis offspring |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111321231B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113699256B (en) * | 2021-09-26 | 2023-06-20 | 浙江省农业科学院 | Functional marker for rapidly identifying gynogenesis offspring of salmon and koi and application of functional marker |
| CN116064824B (en) * | 2022-07-06 | 2024-06-21 | 中国水产科学研究院黑龙江水产研究所 | High-temperature-resistant SNP molecular marker for rainbow trout and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101597648A (en) * | 2009-06-26 | 2009-12-09 | 集美大学 | The screening method of meitogynogenetic diploid of pseudosciaena crocea |
| CN102134593A (en) * | 2010-12-23 | 2011-07-27 | 中国水产科学研究院黄海水产研究所 | Gender-specific microsatellite marker for Cynoglossus semilaevis and application of same in identification of superfemale Cynoglossus semilaevis |
| CN102783445A (en) * | 2012-08-31 | 2012-11-21 | 北京卧佛山庄养殖有限公司 | Method for breeding full-feminization rainbow trout |
| CN103993081A (en) * | 2014-05-16 | 2014-08-20 | 中国水产科学研究院珠江水产研究所 | Method for distinguishing gynogenetic ctenopharyngodon idellus from common ctenopharyngodon idellus |
-
2020
- 2020-03-14 CN CN202010177529.6A patent/CN111321231B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101597648A (en) * | 2009-06-26 | 2009-12-09 | 集美大学 | The screening method of meitogynogenetic diploid of pseudosciaena crocea |
| CN102134593A (en) * | 2010-12-23 | 2011-07-27 | 中国水产科学研究院黄海水产研究所 | Gender-specific microsatellite marker for Cynoglossus semilaevis and application of same in identification of superfemale Cynoglossus semilaevis |
| CN102783445A (en) * | 2012-08-31 | 2012-11-21 | 北京卧佛山庄养殖有限公司 | Method for breeding full-feminization rainbow trout |
| CN103993081A (en) * | 2014-05-16 | 2014-08-20 | 中国水产科学研究院珠江水产研究所 | Method for distinguishing gynogenetic ctenopharyngodon idellus from common ctenopharyngodon idellus |
Non-Patent Citations (6)
| Title |
|---|
| Zhiying JIA 等.Genetic diversity and differentiation of masu salmon(Oncorhynchus masou masou) between and within cultured populations inferred from microsatellite DNA analysis.Zoological Research.2012,第33卷(第E3-4期),全文. * |
| 张艳萍 等.甘肃金鳟遗传资源评价及其与日本金鳟和道氏虹鳟遗传分化研究.水生生物学报.2010,第34卷(第02期),全文. * |
| 张艳萍 等.虹鳟3群体遗传变异的RAPD和SSR分析.西北师范大学学报(自然科学版).2011,第47卷(第05期),全文. * |
| 张艳萍 等.虹鳟性别控制技术研究.万方科技成果数据库.2016,全文. * |
| 徐浩 等.4个虹鳟养殖品系的分子鉴定和遗传多样性分析.中国水产科学.2013,第20卷(第04期),全文. * |
| 杨濯羽 等.基于微卫星标记的22个道氏虹鳟选育家系的遗传多样性研究.淡水渔业.2016,第46卷(第03期),全文. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111321231A (en) | 2020-06-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102134593B (en) | Gender-specific microsatellite marker for Cynoglossus semilaevis and application of same in identification of superfemale Cynoglossus semilaevis | |
| CN106399293B (en) | High-efficiency induction and microsatellite molecular marker identification method for nile tilapia pseudofemale fish | |
| CN111321231B (en) | Method for molecular identification of rainbow trout gynogenesis offspring | |
| CN107217099B (en) | A kind of SNP marker and its application can be used for snakehead genetic sex and the identification of supermale fish | |
| CN106011300A (en) | PCR primer pair used for identifying genetic sex of Pseudobagrus ussuriensis, and rapid identification method thereof | |
| CN115679004B (en) | Primer, method and kit for identifying pseudobagrus vachelli, leiocassis longirostris and hybrid species | |
| CN114657264B (en) | Clarias fuscus sex-specific molecular marker primer and application thereof | |
| CN113862379B (en) | Leiocassis longirostris male sex specific molecular marker, amplification primer thereof and genetic sex identification method | |
| CN105755116B (en) | Primer and its application with microsatellite marker mutually chain whether Eriocheir sinensis sex premature | |
| CN111057771B (en) | SNP molecular marker for distinguishing 'Zhongyang No. 1' from common fugu obscurus and application thereof | |
| CN113151430A (en) | Penaeus chinensis female specific primer and application thereof | |
| CN116356006A (en) | Molecular marker for identifying genetic sex of black carp and application thereof | |
| CN113699224B (en) | Molecular marker C2 for sex identification of Chinese prawn and application thereof | |
| CN113249497B (en) | SNP molecular marker related to growth traits of mandarin fish, primer and application | |
| CN102134600B (en) | PCR (Polymerase Chain Reaction) method for sex appraisal of Nipponia nippon | |
| CN111748639A (en) | Molecular marker for identifying sex of haliotis discus hannai and application thereof | |
| CN104630210B (en) | GLI2 genes are used as pig birth weight proterties related molecular marker and preparation method and application | |
| CN110724750B (en) | Molecular marker C242 of portunus trituberculatus parahaemolyticus and application thereof | |
| CN112458183B (en) | Copy number variation molecular marker on pig No. 3 chromosome and related to daily gain and weight and age on market of pig and application thereof | |
| CN114921566A (en) | Molecular marker for identifying pengze crucian inbred progeny and elopichthys bambusa induced allopetraploid crucian gynogenesis progeny and kit thereof | |
| CN118374586B (en) | Sex identification method for verasper variegatus | |
| CN117487931B (en) | Sillago sihama hypoxia tolerance character related SNP molecular marker and application thereof | |
| CN116875707A (en) | Primer for identifying pelteobagrus fulvidraco and pelteobagrus vachelli, and method and application thereof | |
| CN118638910A (en) | PCR (polymerase chain reaction) primer for sex identification of mystus and identification method | |
| CN104293972B (en) | A kind of differentiate hickie torgoch and brook trout sashimi and the molecular labeling of bacon goods and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |