Primers, kits and identification methods for identifying individuals of Lhasa schizothorax released for reproduction

Abstract

The present invention belongs to the field of animal molecular biology technology and relates to primers and applications for identifying Lhasa schizothorax. The present invention provides primers and applications for accurately assessing the effectiveness of Lhasa schizothorax release. The primers are highly feasible and accurate, and can be widely applied to assess the effectiveness of Lhasa schizothorax reproduction and release.

Description

Technical Field

[0001] The invention belongs to the technical field of animal molecular biology and relates to a primer, a kit and an identification method for identifying individuals of the multiplied and released Lhasa schizothorax. Background Art

[0002] The Lhasa schizothorax (Schizothorax waltoni) belongs to the Cypriniformes, Cyprinidae, subfamily Schizothoracinae, and genus Schizothorax. It is found only in the middle and upper reaches of the Yarlung Zangbo River, its tributaries, and associated waters in the Tibet Autonomous Region, making it endemic to my country. It belongs to the Cyprinidae family in the order Cypriniformes. It is named for the large scales arranged in rows on either side of its anal fin, which resemble abdominal slits between the scales. The genus Schizothorax (Tibetan Schizothorax) comprises 15 species, representing 38.5% of the 39 known species in China. These species are primarily found in the relatively low-elevation reaches of the Jinsha, Lancang, Nu, Irrawaddy, and lower reaches of the Yarlung Zangbo Rivers. Only the Lhasa Schizothorax, the giant beard Schizothorax, the heterodontos, the horizontal mouth Schizothorax, and the Tibetan Schizothorax are found at elevations of 3,000 to 4,500 meters in the middle reaches of the Yarlung Zangbo River, the upper Indus River, and Pangong Lake, representing the highest elevations of the genus. Analysis of their habitats reveals that, with the exception of the Tibetan Schizothorax, which can be found in both rivers and lakes, the others are found in outflowing rivers.

[0003] Stocking is the artificial release of aquatic organisms, whether seedlings or broodstock, into public waters such as oceans, rivers, and lakes. Stocking can replenish and restore biological populations. Due to declining aquatic biomass, or even depletion of some resources, stocking can artificially replenish these resources, thereby increasing resources, improving population structure, and maintaining biodiversity. Stocking can also help protect endangered species, such as those currently rare and protected species, by increasing their numbers through stocking.

[0004] It is an indisputable fact that the number of Schizothorax waltoni has decreased sharply. In order to better protect the wild resources of Schizothorax waltoni, the activity of releasing Schizothorax waltoni for proliferation has been carried out for many consecutive years, making great contributions to the protection of Schizothorax waltoni. With the mature development of the activity of releasing Schizothorax waltoni for proliferation, the evaluation of the release effect of Schizothorax waltoni has received more and more attention. The traditional method for evaluating the release effect is to analyze and evaluate the survival and growth status of the released fish in the released river section through means such as fishing collection, information investigation and data collection, and to track and monitor the effect of the release for proliferation. The specific methods of traditional release are fin-clipping method, tagging method, in-body standard method, etc. The above methods are simple to operate and do not require instruments. However, the above methods also have disadvantages. For example, they will affect the swimming speed and survival of the released individuals. The tags of the tagging method are easy to fall off, and the in-body standard method is expensive, etc. In recent years, microsatellite molecular markers, as a new type of marking method, have been gradually tried at home and abroad. Compared with the traditional marking method, the molecular marker technology is a genetic marker based on the genetic material between individuals, and there is no situation of falling off or changing, which minimizes the harm caused by the traditional marking method to the fish body and is suitable for the evaluation of the release activity effect of fish. Summary of the Invention

[0005] In order to solve the above technical problems existing in the background technology, the present invention provides a primer and an application kit for identifying Schizothorax waltoni, which can accurately evaluate the release effect of Schizothorax waltoni.

[0006] In order to achieve the above purpose, the present invention adopts the following technical scheme:

[0007] A primer for identifying the individuals released for the proliferation of Schizothorax waltoni, characterized in that: the primer for identifying Schizothorax waltoni includes 10 pairs of primers, and the sequences and annealing temperatures of the 10 pairs of primers are respectively:

[0008]

[0009] Preferably, among the primers for identifying the individuals released for the proliferation of Schizothorax waltoni provided by the present invention, primer LSLFY1 and primer LSLFY2 are used in combination, and the two pairs of primers are simultaneously subjected to PCR amplification in the same centrifuge tube; primer LSLFY3 and primer LSLFY4 are used in combination, and the two pairs of primers are simultaneously subjected to PCR amplification in the same centrifuge tube; primer LSLFY5 and primer LSLFY6 are used in combination, and the two pairs of primers are simultaneously subjected to PCR amplification in the same centrifuge tube; primer LSLFY7 and primer LSLFY8 are used in combination, and the two pairs of primers are simultaneously subjected to PCR amplification in the same centrifuge tube; primer LSLFY9 and primer LSLFY10 are used in combination, and the two pairs of primers are simultaneously subjected to PCR amplification in the same centrifuge tube.

[0010] Preferably, the primers provided by the present invention for identifying individuals of the multiplied and released Lhasa schizothorax are used in identifying Lhasa schizothorax.

[0011] Preferably, the primers provided by the present invention for identifying individuals of the multiplied and released Lhasa schizothorax are used in parentage testing of Lhasa schizothorax.

[0012] Preferably, the primers provided by the present invention for identifying individuals of the Lhasa schizothorax for reproduction and release are used in evaluating the effect of releasing the Lhasa schizothorax.

[0013] A detection kit is prepared based on the primers described above for identifying individuals of the Lhasa schizothorax that have been released for reproduction.

[0014] A detection kit for identifying Lhasa schizothorax, characterized in that the detection kit for identifying Lhasa schizothorax includes 10×PCRBuffer, 2.5mmol / L dNTP, 2mmol / L MgCl2, 0.5U / μL Taq enzyme, ultrapure water and primers for identifying Lhasa schizothorax proliferation and release individuals as described above.

[0015] A method for evaluating the effect of the reproduction and release of Lhasa schizothorax, characterized in that the method for evaluating the effect of the reproduction and release of Lhasa schizothorax comprises the following steps:

[0016] 1) Preserving DNA of artificially bred Lhasa schizothorax parents 亲本 ;

[0017] 2) releasing the artificially bred offspring of Lhasa schizothorax;

[0018] 3) fishing for wild populations of Lhasa schizothoracis;

[0019] 4) Extract DNA from wild-caught Lhasa schizothorax 捕捞 ;

[0020] 5) using the aforementioned primers for identifying the Lhasa Schizothorax proliferated and released individuals or the aforementioned detection kit for identifying the Lhasa Schizothorax to detect the DNA obtained in step 1) 亲本 and the DNA obtained in step 4) 捕捞 PCR amplification was performed, and the PCR products were subjected to polyacrylamide gel electrophoresis;

[0021] 6) Analyze the parent-offspring relationship of the Lhasa schizothorax parent and the Lhasa schizothorax caught in the wild based on the results of step 5), obtain the number of Lhasa schizothorax parents in the caught Lhasa schizothorax, and evaluate the release effect based on the number.

[0022] Preferably, the reaction system for PCR amplification in step 5) of the present invention is: 25 μL: 3 μL of 10×PCR Buffer, 2 μL of 2.5 mmol / L dNTP, 3 μL of 2 mmol / L MgCl2, 1 μL each of the upstream and downstream primers, 0.5 μL of 0.5 U / μL Taq enzyme, 2 μL of DNA template, and 10.5 μL of ultrapure water;

[0023] The procedure for the PCR amplification is: pre-denaturation at 94 °C for 3 min; denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 29 s, for 35 cycles; extension at 72 °C for 10 min; preservation at 4 °C.

[0024] Preferably, the method for identifying the parent-child relationship in step 6) of the present invention is: judging according to the electrophoresis result whether all the amplified alleles using the primers for identifying the released individuals of Schizothorax waltoni or the detection kit for identifying Schizothorax waltoni come from the Schizothorax waltoni parents. If so, the wild-caught Schizothorax waltoni individuals have a parent-child relationship with the Schizothorax waltoni in step 2); if not, they do not have a parent-child relationship.

[0025] The present invention has the following advantages:

[0026] The present invention can accurately identify whether the wild Schizothorax waltoni is an individual of the released Schizothorax waltoni with 100% accuracy. The operation of evaluating the release effect of the present invention is simple, and only the DNA samples of the parent individuals need to be preserved. The present invention does not require sampling or tagging the released individuals, avoiding the problem of difficult swimming of the released individuals in the wild environment. Description of the Drawings

[0027] Figure 1 It is the gel map of primers LSLFY1 and LSLFY2 in 48 offspring. Detailed Embodiments

[0028] Example 1 Obtaining the DNA of Schizothorax waltoni

[0029] 1) Take a total of 100 fin rays of the male and female parents of Schizothorax waltoni and their offspring, and preserve them in 95% alcohol. Take out the fin ray samples of each fish from 95% alcohol, cut off about 50 mg, wash them twice in two 500 ml beakers filled with deionized water, and then put them into 1.5 ml centrifuge tubes, and number the centrifuge tubes with a marker pen;

[0030] 2) Add 500 μl of TE extraction buffer to the 1.5 ml centrifuge tube containing the rinsed fin ray tissue, and then start to cut the tissue until there is no large piece of tissue in the centrifuge tube;

[0031] 3) After cutting the tissue into pieces, add 50 μl of 10% SDS solution and 5 μl of proteinase K. Mix well and place in a water bath at 56 °C for 3 - 4 hours until the solution in the centrifuge tube becomes clear;

[0032] 4) During the water bath, shake the centrifuge tube every half hour to ensure thorough mixing and reaction;

[0033] 5) After the solution in the centrifuge tube becomes clear, take it out of the water bath and perform the following operations in the fume hood. Add 500 μl of phenol / chloroform / isoamyl alcohol mixed solution (25:24:1) to the centrifuge tube. Note that the upper layer of the phenol / chloroform / isoamyl alcohol solution is the protective liquid, and the lower layer is the liquid required for the experiment. The pipette tip must be inserted into the lower layer to aspirate the liquid. Shake gently for 8 minutes (try to be as gentle as possible), then place it in a low-temperature high-speed centrifuge and set the centrifugation conditions as follows: centrifuge at 4 °C, 12000 r / min for 10 minutes;

[0034] 6) After centrifugation, use a 100 μl pipette to aspirate the supernatant, then add an equal volume of phenol / chloroform / isoamyl alcohol mixed solution, shake gently for 8 minutes, place it in a low-temperature high-speed centrifuge, and set the conditions as follows: centrifuge at 4 °C, 12000 r / min for 10 minutes:

[0035] 7) After centrifugation, take out the centrifuge tube, use a 100 μl pipette to aspirate the supernatant, then add an equal volume of chloroform / isoamyl alcohol (24:1) solution, shake for 8 minutes, and then centrifuge at 4 °C, 12000 r / min for 10 minutes;

[0036] 8) Take the supernatant, add 1 / 25 volume of 5 M NaCl solution, then add 2 volumes of frozen absolute ethanol, shake well, place in a -20 °C refrigerator, and let it stand for 30 minutes;

[0037] 9) Take out the centrifuge tube, shake out the precipitate by hand, and then centrifuge at 4 °C, 12000 r / min for 10 minutes;

[0038] 10) Discard the supernatant, add 500 μl of 70% alcohol, let it stand for 10 minutes, and then centrifuge at 4 °C, 12000 r / min for 10 minutes;

[0039] 11) Discard the supernatant, and place the centrifuge tube containing the DNA precipitate in the fume hood to dry naturally;

[0040] 12) Add 100 μl of TE to dissolve the template in a 1.5 ml centrifuge tube.

[0041] 13) Let it stand for a while, measure the DNA concentration, and then store it in a 4 °C refrigerator for later use.

[0042] Example 2 Application of microsatellite markers of Schizothorax waltoni in paternity analysis

[0043] Using the extracted DNA as a template, PCR amplification was performed with the microsatellite marker combination of the present invention (the primer sequences are shown in the following table). The PCR amplification reaction system was: 25 μL: 3 μL of 10×PCR Buffer, 2 μL of 2.5 mmol / L dNTP, 3 μL of 2 mmol / L MgCl2, 1 μL each of the upstream and downstream primers, 0.5 μL of 0.5 U / μL Taq enzyme, 2 μL of DNA template, and 10.5 μL of ultrapure water. The PCR amplification program was: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 29 s, for 35 cycles; extension at 72°C for 10 min; and preservation at 4°C.

[0044] The PCR products were electrophoresed in a 10% polyacrylamide gel, and the band results shown by the electrophoresis were analyzed to detect whether each band of the offspring, that is, the allele, came from the parent. The experimental results are shown in the following table. Figure 1 It is the gel diagram of primers LSLFY1 and LSLFY2 in 48 offspring. From Figure 1 it can be seen that the bands amplified by these two pairs of primers in 48 individuals are clear and have high polymorphism. They are excellent loci and can be applied to the paternity analysis of Schizothorax waltoni.

[0045]

[0046]

[0047]

[0048]

[0049] As can be seen from the above table, the microsatellite markers of this patent can accurately perform paternity testing of Schizothorax waltoni, with an accuracy rate of 100%.

[0050] Example 3 Evaluation of Release Effect

[0051] In April 2023, more than 30,000 offspring of Schizothorax waltoni were bred in the Schizothorax waltoni farm, and the DNA information of the parents was well preserved. 30,000 offspring of Schizothorax waltoni were released at the Schizothorax waltoni release point. Half a year after the release, 430 Schizothorax waltoni were caught in the local river. The DNA of each caught Schizothorax waltoni was extracted, and the paternity relationship between the parent DNA and the caught Schizothorax waltoni DNA was identified and analyzed using this paternity testing method. The specific method was as follows:

[0052] The DNA of the parent obtained in step 1) and the DNA of the captured Lhasa schizothorax were amplified by PCR using primers for identifying individuals of the fish released for reproduction or a detection kit for identifying Lhasa schizothorax, and the PCR products were subjected to polyacrylamide gel electrophoresis; wherein, the reaction system for PCR amplification is: 25 μL: 3 μL of 10×PCR Buffer, 2 μL of 2.5 mmol / L dNTP, 3 μL of 2 mmol / L MgCl2, 1 μL of each upstream and downstream primer, 0.5 μL of 0.5 U / μL Taq enzyme, 2 μL of DNA template, and 10.5 μL of ultrapure water; the PCR amplification procedure is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 29 s, 35 cycles; extension at 72°C for 10 min; and storage at 4°C.

[0053] Based on the electrophoresis results, it is determined whether the alleles amplified from the captured Lhasa schizothorax DNA are all from the Lhasa schizothorax parents in the farm. If so, the Lhasa schizothorax individuals caught in the wild have a parent-offspring relationship with the Lhasa schizothorax parents in the farm; if not, they do not have a parent-offspring relationship.

[0054] According to the experimental results, the parent-offspring relationship of the Lhasa schizothorax parents and the Lhasa schizothorax caught in the wild was analyzed. The experimental results showed that among the 430 Lhasa schizothorax caught, the number of released Lhasa schizothorax offspring was 56, and the number of released Lhasa schizothorax accounted for 13% of the local Lhasa schizothorax population, indicating that the release of Lhasa schizothorax was effective.

[0055] The present invention can be applied to the evaluation of the effect of releasing Lhasa schizothorax.

Claims

1. A primer for identifying individuals of Lhasa schizothorax for proliferation and release, characterized in that: The primers for identifying the proliferation and release of Lhasa schizothorax individuals include 10 pairs of primers, and the sequences and annealing temperatures of the 10 pairs of primers are:

2. The primer for identifying individuals of Lhasa schizothorax for proliferation and release according to claim 1, characterized in that: Among the primers used to identify the Lhasa schizothorax proliferation and release individuals, primer LSLFY1 is used in combination with primer LSLFY2; primer LSLFY3 is used in combination with primer LSLFY4; primer LSLFY5 is used in combination with primer LSLFY6; primer LSLFY7 is used in combination with primer LSLFY8; and primer LSLFY9 is used in combination with primer LSLFY10.

3. The use of the primers for identifying the Lhasa schizothorax for proliferation and release as claimed in claim 1 or 2 in identifying the Lhasa schizothorax.

4. The use of the primers for identifying the Lhasa schizothorax proliferation and release individuals as described in claim 1 or 2 in performing Lhasa schizothorax paternity testing.

5. Use of the primers for identifying individuals of Lhasa schizothorax for reproduction and release as described in claim 1 or 2 in evaluating the effect of releasing Lhasa schizothorax.

6. A detection kit prepared based on the primers for identifying individuals of the Lhasa schizothorax for reproduction and release as described in claim 1 or 2.

7. A detection kit for identifying Schizothorax waltoni, characterized in that: The detection kit for identifying Lhasa schizothorax comprises 10×PCR buffer, 2.5 mmol / L dNTP, 2 mmol / L MgCl2, 0.5 U / μL Taq enzyme, ultrapure water, and the primers for identifying Lhasa schizothorax proliferation and release individuals as described in claim 1 or 2.

8. A method for evaluating the effect of stocking and restocking of Lhasa schizothorax, characterized in that: The method for evaluating the effect of the proliferation and release of Lhasa schizothorax comprises the following steps: 1) Preserve the DNA of the parental fish for artificial breeding of Schizothorax waltoni 亲本 ; 2) releasing the artificially bred offspring of Lhasa schizothorax; 3) fishing for wild populations of Lhasa schizothoracis; 4) Extract the DNA of Schizothorax waltoni captured from the wild population 捕捞 ; 5) using the primers for identifying the Lhasa schizothorax proliferation and release individuals according to claim 1 or the detection kit for identifying the Lhasa schizothorax according to claim 7 to detect the DNA obtained in step 1) 亲本 and the DNA obtained in step 4) 捕捞 PCR amplification was performed, and the PCR products were subjected to polyacrylamide gel electrophoresis; 6) Analyze the parent-offspring relationship of the Lhasa schizothorax parent and the Lhasa schizothorax caught in the wild based on the results of step 5), obtain the number of Lhasa schizothorax parents in the caught Lhasa schizothorax, and evaluate the release effect based on the number.

9. The method for evaluating the effect of proliferation and release of Lhasa schizothorax according to claim 8, characterized in that: The reaction system for PCR amplification in step 5) is as follows: 25 μL: 3 μL of 10×PCR Buffer, 2 μL of 2.5 mmol / L dNTPs, 3 μL of 2 mmol / L MgCl, 1 μL of each of upstream and downstream primers, 0.5 μL of 0.5 U / μL Taq enzyme, 2 μL of DNA template, and 10.5 μL of ultrapure water; The PCR amplification procedure is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 29 s, 35 cycles; extension at 72°C for 10 min; and storage at 4°C.

10. The method for evaluating the effect of proliferation and release of Lhasa schizothorax according to claim 8 or 9, characterized in that: The method for identifying the parent-offspring relationship in step 6) is: judging whether the alleles amplified by the primers for identifying the Lhasa schizothorax propagation and release individuals described in claim 1 or the detection kit for identifying the Lhasa schizothorax described in claim 7 are all from the Lhasa schizothorax parent based on the electrophoresis results; if so, the Lhasa schizothorax individual caught in the wild has a parent-offspring relationship with the Lhasa schizothorax in step 2); if not, there is no parent-offspring relationship.

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