CN113481302B - Catfish marking releasing effect evaluation method - Google Patents
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- CN113481302B CN113481302B CN202110496891.4A CN202110496891A CN113481302B CN 113481302 B CN113481302 B CN 113481302B CN 202110496891 A CN202110496891 A CN 202110496891A CN 113481302 B CN113481302 B CN 113481302B
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention discloses a catfish marking releasing effect evaluation method, which specifically comprises the following steps: dividing the released catfish into large-individual catfish and small-individual catfish, implanting microsatellite markers in abdominal cavities of the large-individual catfish, and fixing external hanging markers on dorsal fins of the small-individual catfish; carrying out fin shearing ray sampling on the released catfish and recording the information of the catfish; identifying the genetic information of each catfish by using a microsatellite marking technology, counting the alleles of each released catfish and constructing a sample genetic file; after the catfish is released, a satellite signal receiver is placed at a river fixed-point position, satellite signals are collected, the moving range of the released catfish is determined, and the catfish in the releasing area is recaptured in the catching period; and identifying the recaptured individuals, and counting the number of released individuals in the recaptured catfish. The method can effectively identify the proportion of the released catfish in the releasing water area, so as to evaluate the releasing effect of the catfish, and has great popularization value.
Description
Technical Field
The invention belongs to the technical field of proliferation and releasing, and particularly relates to a catfish marking and releasing effect evaluation method.
Background
With the increase of the range of human activities, the living environment of fishes is greatly affected, and the species and the number of fishes are sharply decreased. The proliferation and releasing is to release aquatic fry and parents to public water areas, such as sea, river, lake, etc. manually. The population of biological resources can be supplemented and recovered through proliferation and releasing, because the aquatic resources are reduced year by year, and some biological resources are even few or none, and the biological resources are artificially supplemented into water through proliferation and releasing, so that the resources are increased, the population structure of organisms is improved, and meanwhile, the diversity of the organisms can be maintained. For some endangered species, the number of the endangered species can be increased by means of proliferation and releasing, and the protection effect on the endangered species is achieved.
Catfish are typical herbivorous fish, inhabit rivers and lakes in plain areas, and are generally favored in the middle and lower layers of water and near-shore multi-aquatic areas. It is lively, swims rapidly, and often finds food in groups. However, as water resources are destroyed, the number and size of catfish are gradually reduced. Manual releasing of catfishes is also imminent. However, the effect of releasing the catfish is not reported at present, and a method for effectively evaluating the artificial releasing effect of the catfish is urgently needed.
Disclosure of Invention
The invention aims to provide a catfish mark releasing effect evaluation method, which improves the catfish proliferation and releasing efficiency.
The technical scheme adopted by the invention is a catfish marking releasing effect evaluation method, which specifically comprises the following steps:
step 1, marking; the method divides the released catfish into two groups, wherein the catfish with the weight of more than 10 kg is divided into one group, and the catfish with the weight of less than 10 kg is divided into one group. Implanting satellite markers into abdominal cavity of large individual catfish, and fixing external hanging markers on dorsal fins of small individual catfish;
step 2, collecting a sample; cutting fin lines of the catfishes released every year, sampling and recording the age, the weight, the shape and the like of the catfishes so as to know the field growth of the catfishes in the future;
and step 3: constructing a sample genetic file; identifying the genetic information of each catfish by using a microsatellite marking technology, counting the alleles of each released catfish and constructing a sample genetic file; supplementing released catfish genetic information into a sample genetic archive before artificial catfish proliferation and releasing every year;
step 4, capturing the released population; after the catfish is released, a satellite signal receiver is placed at a river fixed-point position, satellite signals are collected, the moving range of the released catfish is determined, and the catfish in the releasing area is recaptured in the catching period;
and 5, identifying the recaptured individuals, and counting the number of the recaptured catfishes as released individuals. The specific statistical method is to check whether the dorsal fin has the external hanging mark and check whether the abdomen of the fish has an incision and a stitching trace left when the satellite is placed. If neither of the two are available, the individual identification of the recaptured catfish is carried out by using a microsatellite marking technology. Calculating the proportion of released individuals in the recapture population, wherein the formula for calculating the recapture rate is as follows: (the number of released groups in the recaptured samples/the total number of the recaptured samples)%, the larger the recapture rate is, the better the proliferation and release effect is, so that the contribution of catfish proliferation and release to all the ecological quantities of catfish in Yangtze river is estimated, and the catfish proliferation and release effect is further determined.
The present invention is also characterized in that,
in step 3, constructing a sample genetic file, specifically:
step 3.1, taking all released catfish fin line samples, extracting catfish sample DNA, and performing PCR amplification on the catfish sample by using 5 groups of double PCR consisting of 10 pairs of microsatellite primers; obtaining a PCR amplification product;
and 3.2, performing electrophoretic separation on the PCR amplification product on 10 volume percent polyacrylamide gel, and counting the genotype of the PCR amplification product according to the separation result.
The PCR reaction system was 25ul:10 XPCR Buffer 3ul, 2.5mmol/L dNTP 2ul, MgCl23ul, 1ul of each of the upstream and downstream primers, 0.5ul of Taq enzyme, 2ul of DNA template, and 10.5ul of ultrapure water.
The PCR reaction program is: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing temperature renaturation for 30s, extension at 72 ℃ for 30s, and 30 cycles; extending for 10min at 72 ℃; storing at 4 ℃.
10 pairs of catfish microsatellite primers, wherein the specific primer information is as follows:
the specific method for carrying out individual identification on the recaptured catfish by utilizing the microsatellite marker technology comprises the following steps: extracting DNA of the captured catfish; forming 5 groups of double PCR by using 10 pairs of catfish microsatellite primers to carry out PCR amplification on the catfish sample; carrying out electrophoretic separation on the PCR amplification product on 10% polyacrylamide gel; counting the genotype of the PCR amplification product according to the separation result; and comparing the obtained genotype with the sample genetic file to determine the number of released individuals in the caught individuals.
The invention has the beneficial effects that:
the method for evaluating the releasing effect of the catfish marker can effectively identify the proportion of released catfish in a releasing water area, further evaluate the releasing effect of the catfish, and has great popularization value.
Detailed Description
The present invention will be described in detail with reference to the following embodiments.
The invention relates to a catfish marking releasing effect evaluation method, which specifically comprises the following steps:
step 1, marking; dividing the released catfish into large-individual catfish and small-individual catfish, implanting microsatellite markers in abdominal cavities of the large-individual catfish, and fixing external hanging markers on dorsal fins of the small-individual catfish;
the weight of the large individual catfish is more than or equal to 10 kilograms, and the weight of the small individual catfish is less than 10 kilograms;
step 2, collecting a sample; carrying out fin shearing ray sampling on the released catfish and recording the information of the catfish;
the information of the catfish comprises age, weight, size, shape and the like so as to know the field growth of the catfish in the future;
and step 3: constructing a sample genetic file; identifying the genetic information of each catfish by using a microsatellite marking technology, counting the alleles of each released catfish and constructing a sample genetic file; supplementing released catfish genetic information into a sample genetic archive before artificial catfish proliferation and releasing every year;
constructing a sample genetic file, which specifically comprises the following steps:
step 3.1, taking all released catfish fin line samples, extracting catfish sample DNA, and performing PCR amplification on the catfish sample by using 10 pairs of microsatellite primers; obtaining a PCR amplification product;
the PCR reaction system was 25ul:10 XPCR Buffer 3ul, 2.5mmol/L dNTP 2ul, MgCl23ul of primers at the upstream and downstream, 1ul of each primer, 0.5ul of Taq enzyme, 2ul of DNA template and 10.5ul of ultrapure water;
the PCR reaction program is: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing temperature renaturation for 30s, extension at 72 ℃ for 30s, and 30 cycles; extending for 10min at 72 ℃; storing at 4 deg.C;
10 pairs of catfish microsatellite primers form 5 groups of double PCR, and the specific primer information is as follows:
and 3.2, performing electrophoretic separation on the PCR amplification product on 10 volume percent polyacrylamide gel, and counting the genotype of the PCR amplification product according to the separation result.
Step 4, capturing the released population; after the catfish is released, a satellite signal receiver is placed at a river fixed-point position, satellite signals are collected, the moving range of the released catfish is determined, and the catfish in the releasing area is recaptured in the catching period;
step 5, identifying the recaptured individuals, and counting the number of released individuals in the recaptured catfishes;
the specific statistical method comprises the following steps: checking whether the dorsal fin of the catfish has an external hanging mark, and checking whether the abdomen of the catfish has an incision and a stitching trace left when a satellite is placed; if the two are not available, the individual identification of the recaptured catfish is carried out by utilizing the microsatellite marking technology; calculating the proportion of the released individuals in the recapture population, wherein the formula for calculating the recapture rate is as follows: (the number of released groups in the recaptured samples/the total number of the recaptured samples)%, the larger the recapture rate is, the better the proliferation and release effect is, so that the contribution of catfish proliferation and release to all the ecological quantities of catfish in Yangtze river is estimated, and the catfish proliferation and release effect is further determined.
The specific method for carrying out individual identification on the recaptured catfish by utilizing the microsatellite marker technology comprises the following steps: extracting DNA of the captured catfish; performing PCR amplification on the catfish sample by using the 10 pairs of catfish microsatellite marker primers provided in the step 3; carrying out electrophoretic separation on the PCR amplification product on 10% polyacrylamide gel; counting the genotype of the PCR amplification product according to the separation result; and comparing the obtained genotype with the sample genetic file to determine the number of released individuals in the caught individuals.
Examples
The released catfishes are divided into two groups, wherein the catfishes more than 10 kg are divided into one group, and the catfishes less than 10 kg are divided into one group. The satellite mark is implanted into the abdominal cavity of the large-individual catfish, and the external hanging mark is fixed on the dorsal fin of the small-individual catfish. The yearly released catfish is sampled by cutting fin line and the age, weight, shape and the like of the catfish are recorded so as to know the field growth of the catfish in the future. And identifying the genetic information of each catfish by using a microsatellite marking technology, counting the alleles of each released catfish and constructing a sample genetic file. The released catfish genetic information is supplemented into a sample genetic archive before the artificial catfish proliferation and releasing are carried out every year. After releasing the catfish, a satellite signal receiver is placed at a fixed-point position of a river, satellite signals are collected, and the moving range of the released fish is determined. And fishing the catfish in the drainage area in the fishing period. Identifying the recaptured individuals, and counting the number of released individuals in the recaptured catfishes. The specific statistical method is to check whether the dorsal fin has the external hanging mark and check whether the abdomen of the fish has an incision and a stitching trace left when the satellite is placed. If neither of the two are available, the individual identification of the recaptured catfish is carried out by using a microsatellite marking technology. Calculating the proportion of released individuals in the recapture population, wherein the formula for calculating the recapture rate is as follows: (the number of released groups in the recapture samples/the total number of recaptured samples)%, the larger the recapture rate is, the better the proliferation and release effects are, so that the contribution of catfish proliferation and release to all the ecological quantities of catfishes in Yangtze river is estimated, and the catfish proliferation and release effects are further determined.
Establishing a sample genetic file, which specifically comprises the following steps:
extracting DNA of all fish to be released, specifically comprising:
(1) taking out the stored fin ray sample, shearing about 0.5g, putting into a 2mL sterile centrifuge tube, adding 1 XTE sterile buffer solution, and soaking at 4 ℃ for 12 h;
(2) taking out the fin-shaped tissue, putting into 352ul sterile extraction buffer, gently mixing, adding 40ul 20% SDS and 10ul 20mg/mL proteinase K (final concentration is 400ug/mL), and mixing;
(3) incubate at 56 ℃ for 4h, gently shake the tube every half hour to facilitate digestion. If the tissue is not digestible, the incubation time can be prolonged to 8h, or digestion is carried out overnight at 37 ℃:
(4) after the tissues are completely digested, taking out the centrifuge tube, adding 300ul of 6M NaCI solution, and immediately and violently shaking for 30S;
(5) centrifuging at 12000rpm for 10 min;
(6) discarding the precipitate, pouring the supernatant into a new centrifuge tube, centrifuging once under the same conditions, taking the supernatant, adding equal volume of pre-cooled isopropanol, and standing at 20 ℃ for more than 1 h;
(7) centrifuging at 12000rpm for 15min at 4 deg.C;
(8) discarding the supernatant, washing the precipitate with 70% ethanol solution once, and oven drying at 37 deg.C or naturally air drying;
(9) 50uL of sterilized double distilled water is added to dissolve DNA, and after complete dissolution, the DNA is diluted to 100ng/uL and stored in a refrigerator at 4 ℃ for later use.
DNA quality detection
The integrity and purity of the DNA are detected by 1% agarose gel electrophoresis, which comprises the following steps:
1) preparing 0.8% agarose gel by using 1xTAE electrophoresis buffer solution, heating the lmin at medium temperature by microwave to dissolve the agarose gel, filling tap water to brush the outer bottle wall to cool down, adding 2ul EB dye solution into a conical flask after the gel is cooled to about 50 ℃, quickly mixing the solution uniformly, and pouring the solution into a clean gel tank to prepare the gel.
2) Adding the 1xTAE electrophoresis buffer solution into a horizontal electrophoresis apparatus in advance, slightly pulling out a comb after the gel is solidified, and putting the agarose gel into the horizontal electrophoresis apparatus to ensure that the electrophoresis buffer solution completely submerges the agarose gel.
3) After 5ul of prepared DNA sample is mixed with 1ul of 6xloading buffer, the mixture is accurately added into the sample well, and the position of gel is prevented from moving in the process.
4) The voltage of the electrophoresis apparatus is set to be 100V, and the electrophoresis time is about 20 min.
5) And after the electrophoresis is finished, the power supply of the electrophoresis apparatus is closed, the agarose gel is taken out, and the agarose gel is placed in a gel imaging system to observe the electrophoresis result.
Screening for microsatellite markers
The catfish DNA extracted in the example is used to screen microsatellite loci with better polymorphism. The PCR reaction system is established with 25ul of 10 XPCR Buffer 3ul, 2.5mmol/L dNTP 2ul, MgCl 23 ul, upstream and downstream primers 1ul, Taq enzyme 1ul, DNA template 2ul and ultrapure water 12 ul. The PCR reaction condition is pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, renaturation for 30s, and denaturation at 72 ℃ for 45s, and 30 cycles; extending for 7min at 72 ℃; the product was stored at 4 ℃. The PCR product was electrophoresed on a 10% polyacrylamide gel for 150 minutes at a voltage of 220V. The polyacrylamide gels were then silver stained and photographed. And (3) selecting microsatellite marker sites with clear bands and high polymorphism in experimental results.
Dual PCR microsatellite marker screening
According to the size of the PCR product in the embodiment 2, two microsatellite markers with different sizes of the PCR product are randomly combined into a group of double PCR reaction system, and the PCR reaction system is established to be 25ul:10 XPCR Buffer 4ul, 2.5mmol/L dNTP 3ul, MgCl 24 ul, 1ul of each of the upstream and downstream primers of the two pairs of primers, 1ul of Taq enzyme, 2ul of DNA template and 7ul of ultrapure water. The PCR reaction condition is pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, renaturation for 30s, and denaturation at 72 ℃ for 45s, and 30 cycles; extending for 7min at 72 ℃; the product was stored at 4 ℃. The PCR product was electrophoresed on a 10% polyacrylamide gel for 150 minutes at a voltage of 220V. The polyacrylamide gels were then silver stained and photographed. And selecting the double PCR reaction combination with clear experimental result and high polymorphism.
Construction of catfish genetic archives
The method provided by the invention is used for constructing the genetic file of the catfish to be released. Taking a fin ray sample of the catfish to be released, and extracting DNA. And respectively carrying out PCR amplification on all catfish DNA by using the screened double PCR labeled primers. And (3) performing polyacrylamide gel electrophoresis on the PCR amplification product. And constructing a released catfish genetic file according to the size of the strip displayed by electrophoresis. The genetic file format is shown in table one, the first row is the code number of each catfish individual in vitro tag, the first column is the microsatellite marker shown in the invention, and the rest are the band size of each catfish under each microsatellite marker, as shown in table 1, the construction of the catfish genetic file is completed.
TABLE 1 genetic profile of catfish
Placement of satellite mark in large-body catfish body
The method comprises the steps of cutting an opening with the length of about 1 cm at the lateral position of the abdominal midline and about 3 cm above the anus of a large-size catfish by using a scalpel, plugging a sterilized satellite mark into the body of the catfish, and then suturing the wound by using a thread. And satellite signal receivers are arranged at the upstream and downstream of the release site, satellite signals are collected, and the movement position of the release individual is observed in real time.
External hanging mark for installing small individual catfish
And installing an external hanging mark in the middle of the dorsal fin of the small catfish to be released, and marking the two-dimensional code of releasing information of the catfish and the contact phone by the external hanging mark. The specific method comprises sterilizing the external tag with 70% alcohol, and driving the external tag into the middle part of the dorsal fin of catfish with a release lance. And registering information of each external tag so that the external tags correspond to the catfish genetic file.
Catfish releasing effect assessment
And (5) fishing the catfish back at the upstream and downstream of the catfish releasing place. Identifying the recaptured individuals, and counting the number of released individuals in the recaptured catfishes. The specific statistical method is to check whether the dorsal fin has the external hanging mark and check whether the abdomen of the fish has an incision and a stitching trace left when the satellite is placed. If neither of the two are available, the individual identification of the recaptured catfish is carried out by using a microsatellite marking technology. Calculating the proportion of released individuals in the recapture population, wherein the formula for calculating the recapture rate is as follows: (the number of released groups in the recaptured samples/the total number of the recaptured samples)%, the larger the recapture rate is, the better the proliferation and release effect is, so that the contribution of catfish proliferation and release to all the ecological quantities of catfish in Yangtze river is estimated, and the catfish proliferation and release effect is further determined.
Catfish releasing effect assessment
1) Preserving DNA of released catfish;
2) and (4) catching the catfish 689 tail in the released water area, and screening out catfish with an external hanging mark and catfish with a satellite mark. Extracting DNA of all the rest catfishes;
3) the double PCR system provided by the invention is utilized to carry out PCR amplification on the DNA of the rest catfish;
4) carrying out electrophoretic separation on the amplification result on polyacrylamide gel;
5) counting the genotype of the PCR amplification product according to the separation result;
6) comparing the genetic files of the catfishes according to the genotypes obtained in the previous step, and determining that the number of released individuals in the caught individuals is 43. In the catfish recapture, the releasing individual accounts for 6.2 percent.
SEQUENCE LISTING (SEQUENCE LISTING)
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Claims (3)
1. A catfish marking releasing effect evaluation method is characterized by comprising the following steps:
step 1, marking; the method divides the released catfish into two groups, wherein satellite markers are implanted into the abdominal cavity of a large individual catfish, and external hanging markers are fixed on the dorsal fins of a small individual catfish;
step 2, collecting a sample; cutting fin lines of the catfishes released every year, sampling and recording the age, the weight, the size and the shape of the catfishes so as to know the field growth of the catfishes in the future;
and 3, step 3: constructing a sample genetic file; identifying the genetic information of each catfish by using a microsatellite marking technology, counting the alleles of each released catfish and constructing a sample genetic file; supplementing released catfish genetic information into a sample genetic archive before artificial catfish proliferation and releasing every year;
constructing a sample genetic file, which specifically comprises the following steps:
step 3.1, taking all released catfish fin line samples, extracting catfish sample DNA, and performing PCR amplification on the catfish sample by using 10 pairs of microsatellite primers; obtaining a PCR amplification product;
step 3.2, performing electrophoretic separation on the PCR amplification product on polyacrylamide gel with the volume percentage of 10%, and counting the genotype of the PCR amplification product according to the separation result;
the 10 pairs of microsatellite primers form 5 groups of double PCR, and the specific primer information is as follows:
step 4, capturing the released population; after the catfish is released, a satellite signal receiver is placed at a river fixed-point position, satellite signals are collected, the moving range of the released catfish is determined, and the catfish in the releasing area is recaptured in the catching period;
step 5, identifying the recaptured individuals, and counting the number of the recaptured catfishes as released individuals; the specific statistical method comprises the steps of checking whether the dorsal fin has an external hanging mark, and checking whether the abdomen of the fish has a cut and a stitching trace left when a satellite is placed; if the two are not available, the individual identification of the recaptured catfish is carried out by utilizing the microsatellite marking technology; calculating the proportion of released individuals in the recapture population, wherein the formula for calculating the recapture rate is as follows: (the number of released groups in the recaptured samples/the total number of the recaptured samples) x 100%, the larger the recapture rate is, the better the proliferation and release effects are, so that the contribution of catfish proliferation and release to all the ecological quantity of catfishes in Yangtze river is estimated, and the catfish proliferation and release effects are further determined.
2. The method as claimed in claim 1, wherein in step 1, the weight of the large catfish is 10 kg or more, and the weight of the small catfish is 10 kg or less.
3. The method for evaluating the mark releasing effect of the catfish according to claim 1, wherein the method for identifying the individual caught catfish by using the microsatellite marking technology comprises the following steps: extracting DNA of the captured catfish; forming 5 groups of double PCR by using 10 pairs of catfish microsatellite primers to carry out PCR amplification on the catfish sample; carrying out electrophoretic separation on the PCR amplification product on 10% polyacrylamide gel; counting the genotype of the PCR amplification product according to the separation result; and comparing the obtained genotype with the sample genetic file to determine the number of released individuals in the caught individuals.
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