Microsatellite primer for identifying genetic relationship of siniperca chuatsi and identification method
Through the design of microfinee primers and appraisal methods, the problem of close relatives in large -eyed artificial breeding was solved, and the efficient identification of the close -up relationship of large -eyed pupa was achieved, guiding breeding to avoid close relative reproduction, and improved the performance and resistance of breeding species.
Patent Information
- Application Number
- CN202410477132.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-04-19
- Publication Date
- 2025-01-28
AI Technical Summary
Inbreeding is serious in artificial breeding of big-eyed mandarin, resulting in germplasm degeneration, growth performance and resistance to disease resistance. An effective method is needed to avoid inbreeding.
A microbrims and corresponding appraisal methods are designed. Through PCR amplification and electrical separation, combined with genetic analysis software for clustering analysis, identifying the affinity of large -eyed crickets, thereby guiding artificial reproduction to avoid close relatives.
The family relationship of large -eyed crickets is identified by molecular means, effectively avoiding the reproduction of close relatives, improving the growth performance of breeding species and anti -inverse disease resistance performance, and reducing the cost of the PCR amplification system.
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Figure CN119372323A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of animal molecular genetics, and in particular to a microsatellite primer and an identification method for identifying the kinship of big-eyed mandarin fish. Background Art
[0002] The big-eyed mandarin fish belongs to the genus Siniperca chuatsi, commonly known as sweet-scented osmanthus fish, white osmanthus, etc. It is widely distributed in the Yangtze River and the water system to its south in China. It is a species endemic to southern China. The big-eyed mandarin fish mainly feeds on fish, followed by shrimps. It grows slower than mandarin fish and prefers to live in mainstream water environments. A parent fish weighing 1.5kg can carry more than 100,000 eggs. The big-eyed mandarin fish was described and classified as a species in the early 20th century. Later, scientific researchers conducted relevant research on its morphological characteristics and classification, biology and breeding.
[0003] In recent years, due to human influence, the wild resources of big-eyed mandarin fish and other species have declined sharply. The artificial breeding of big-eyed mandarin fish also faces huge problems: as the breeding volume of big-eyed mandarin fish increases, the phenomenon of inbreeding becomes more and more obvious, resulting in the degradation of germplasm, and the growth performance, resistance to stress and disease of big-eyed mandarin fish are affected. Therefore, how to effectively avoid inbreeding of big-eyed mandarin fish has become an urgent problem to be solved. Summary of the invention
[0004] In order to effectively avoid inbreeding of big-eyed mandarin fish, the present invention provides a microsatellite primer and an identification method for identifying the kinship of big-eyed mandarin fish.
[0005] The technical solution adopted to achieve the first invention object of the present invention is: a microsatellite primer for identifying the kinship of big-eyed mandarin fish, comprising: a nucleotide sequence shown in SEQ ID NO.1-20.
[0006] The technical solution used to achieve the second invention object of the present invention is: a method for identifying the kinship of big-eyed mandarin fish, comprising the following steps: S1, extracting DNA from samples to be identified; S2, using the microsatellite primers described in the above technical solution to perform PCR amplification on the sample DNA extracted in step S1 to obtain an amplified product; S3, performing electrophoresis separation on a 10% polyacrylamide gel on the amplified product obtained in step S2 to obtain an electrophoresis separation result; S4, counting the genotypes of the PCR amplification products of the samples to be identified according to the electrophoresis separation results obtained in step S3, to obtain the genotype results of the samples to be identified; S5. Perform cluster analysis on the genotype results obtained in step S4 by using genetic analysis software to obtain the results of kinship identification of big-eyed mandarin fish: the sample individuals gathered in the same branch have a close relationship, while the sample individuals gathered in different branches have a distant relationship.
[0007] The beneficial effects of the present invention are as follows: (1) the present invention identifies the genetic relationship of the big-eyed mandarin fish by molecular means, guides the artificial breeding of the big-eyed mandarin fish according to the identification results, and can effectively avoid the phenomenon of inbreeding of the big-eyed mandarin fish; (2) The PCR amplification system of the present invention saves half the cost compared with the traditional PCR system; (3) The method for identifying the kinship of big-eyed mandarin fish of the present invention is used to guide the artificial breeding of big-eyed mandarin fish, and can be used for family identification and parentage identification of big-eyed mandarin fish, which is more labor-saving than the traditional marking method (tagging method) of big-eyed mandarin fish.
[0008] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS
[0009] Attached Figure 1 This is a cluster analysis diagram of Example 1 of the present invention.
[0010] Attached Figure 2 This is a cluster analysis diagram of Example 2 of the present invention. DETAILED DESCRIPTION
[0011] The design process of the microsatellite primers for identifying the genetic relationship of big-eyed mandarin fish of the present invention is as follows: The genome data of Siniperca scherzeri were downloaded from NCBI database. The genome data were analyzed by SSRHunter software. The fragments with microsatellite sequences were screened. 200 pairs of microsatellite sequences with repeat sequences of more than 3 bases were randomly selected. Primer 5.0 software was used to design primers for the above 200 pairs of microsatellites.
[0012] The principles of primer design are as follows: a. The primer sequence should be neither too close nor too far from the core sequence, and should be approximately 50-80 bases away; b. The target fragment (PCR product) is of appropriate size, about 100-400 bp: c. The annealing temperature of the primer sequence is between 50℃-60℃; d. The primer sequence must contain G and C, and the content should be between 40% and 60%.
[0013] During the primer design process, you can adjust the conditions appropriately to screen for suitable primers: Take 6 individual fins of bigeye mandarin fish, use the above 200 pairs of microsatellite primers to perform PCR amplification and electrophoresis on the 6 bigeye mandarin fish, the PCR amplification reaction system is 25ul: 10×PCR Buffer 3ul, 2.5mmol / L dNTP 1ul, MgCl23ul, 1ul of upstream and downstream primers, 1ul of Taq enzyme, 2ul of DNA template, 14ul of ultrapure water; PCR amplification reaction program is: 94℃ pre-denaturation for 3 minutes; then 94℃ denaturation for 30 seconds, 58℃ annealing for 30 seconds, 72℃ extension for 45 seconds, a total of 35 cycles; finally 72℃ extension for 10 minutes, 4℃ storage. Find the microsatellite primers that can amplify PCR products in bigeye mandarin fish species.
[0014] Then, the microsatellite primers were combined and double PCR test was performed. The test method was as follows: the screened microsatellite primers were combined in pairs, and then the combined primer sets were used to perform PCR amplification and electrophoresis on 6 bigeye mandarin fish. The PCR amplification reaction system was 25ul: 10×PCR Buffer 3ul, 2.5mmol / L dNTP 1ul, MgCl23ul, 1ul each of the upstream and downstream primers of the two pairs of primers, Taq enzyme 1ul, DNA template 2ul, ultrapure water 12ul; the PCR amplification reaction procedure was: 94℃ pre-denaturation for 3 minutes; then 94℃ denaturation for 30 seconds, 58℃ annealing for 30 seconds, 72℃ extension for 45 seconds, for a total of 35 cycles; finally 72℃ extension for 10 minutes and storage at 4℃.
[0015] Finally, 5 suitable dual PCR primer sets capable of amplifying bigeye mandarin fish were found, which are the 5 dual PCR systems of the present invention, and their nucleotide sequences are the nucleotide sequences shown in SEQ ID NO.1-20 in the sequence table.
[0016] The specific information of these five sets of dual PCR systems is shown in Table 1: Table 1 Nucleotide sequence information of five duplex PCR systems for identifying the relationship of Siniperca chuatsi .
[0017] In the 5 sets of dual PCR systems shown in Table 1, in set 1: DYG1F corresponds to the nucleotide sequence shown in SEQ ID NO.1; DYG1R corresponds to the nucleotide sequence shown in SEQ ID NO.2; DYG2F corresponds to the nucleotide sequence shown in SEQ ID NO.3; and DYG2R corresponds to the nucleotide sequence shown in SEQ ID NO.4.
[0018] Group 2: DYG3F corresponds to the nucleotide sequence shown in SEQ ID NO.5; DYG3R corresponds to the nucleotide sequence shown in SEQ ID NO.6; DYG4F corresponds to the nucleotide sequence shown in SEQ ID NO.7; DYG4R corresponds to the nucleotide sequence shown in SEQ ID NO.8.
[0019] Group 3: DYG5F corresponds to the nucleotide sequence shown in SEQ ID NO.9; DYG5R corresponds to the nucleotide sequence shown in SEQ ID NO.10; DYG6F corresponds to the nucleotide sequence shown in SEQ ID NO.11; DYG6R corresponds to the nucleotide sequence shown in SEQ ID NO.12.
[0020] Group 4: DYG7F corresponds to the nucleotide sequence shown in SEQ ID NO.13; DYG7R corresponds to the nucleotide sequence shown in SEQ ID NO.14; DYG8F corresponds to the nucleotide sequence shown in SEQ ID NO.15; DYG8R corresponds to the nucleotide sequence shown in SEQ ID NO.16.
[0021] Group 5: DYG9F corresponds to the nucleotide sequence shown in SEQ ID NO.17; DYG9R corresponds to the nucleotide sequence shown in SEQ ID NO.18; DYG10F corresponds to the nucleotide sequence shown in SEQ ID NO.19; DYG10R corresponds to the nucleotide sequence shown in SEQ ID NO.20.
[0022] The above five sets of double PCR systems were used to amplify the DNA of bigeye mandarin fish and then identify their kinship. The specific steps are as shown in S1-S5.
[0023] S1. Extract the DNA of the sample to be identified. The specific operation is as follows: In this experiment, the genomic DNA of the test materials was extracted using the tissue genomic DNA extraction kit of Tiangen Biochemical Technology Co., Ltd. The specific steps are as follows: (1) Take an appropriate amount of fin rays and place them in a sterile centrifuge tube containing 1 mL of 95% alcohol. Cut them into pieces to form a cell suspension, then centrifuge at 10,000 r / min for 1 min. Pour off the supernatant, add 200 μL of buffer GA, and vortex until completely suspended. (2) Add 20 ul of Proteinase K solution, mix well, and place at 56°C overnight to allow complete digestion. Centrifuge briefly to remove water droplets on the inner wall of the tube cap before proceeding to the next step; (3) Add 200 μl of buffer GB, mix thoroughly by inversion, and place at 70°C for 10 min. The solution should become clear and centrifuge briefly to remove water droplets on the inner wall of the tube cap. (4) Add 200 μl of anhydrous ethanol and vortex thoroughly for 15 seconds. Flocculent precipitation may appear at this time. Centrifuge briefly to remove water droplets on the inner wall of the tube cap. (5) Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (the adsorption column is placed in a collection tube), centrifuge at 12000 r / min for 30 sec, discard the waste liquid, and place the adsorption column CB3 back into the collection tube; (6) Add 500 μL of buffer GD to the adsorption column CB3 (check whether anhydrous ethanol has been added before use), centrifuge at 12,000 r / min for 30 sec, discard the waste liquid, and place the adsorption column CB3 in a collection tube; (7) Add 600 μl of rinse solution PW to the adsorption column CB3 (check whether anhydrous ethanol has been added before use), centrifuge at 12,000 r / min for 30 sec, discard the waste liquid, and place the adsorption column CB3 in a collection tube; (8) Repeat step 7; (9) Place the adsorption column CB3 back into the collection tube, centrifuge at 12000 r / min for 2 min, and discard the waste liquid. Leave the adsorption column CB3 at room temperature for several minutes to completely dry the remaining rinse liquid in the adsorption material; (10) Transfer the adsorption column CB3 to a clean centrifuge tube, add 50-200ul of elution buffer TE to the middle part of the adsorption membrane, leave it at room temperature for 2-5 min, centrifuge it at 12000 r / min for 2 min, and collect the solution in a centrifuge tube. To increase the yield of genomic DNA, add the solution obtained by centrifugation to the adsorption column CB3, leave it at room temperature for 2 min, and centrifuge it at 12000 r / min for 2 min; (11) DNA purity and concentration were checked by UV spectrophotometer and agarose gel electrophoresis and stored at -20°C until use.
[0024] S2, using the microsatellite primers of the present invention to perform PCR amplification on the sample DNA extracted in step S1 to obtain an amplified product; The reaction system for PCR amplification is 25ul: 10×PCR Buffer 3ul, 2.5mmol / L dNTP 1ul, MgCl2 3ul, 1ul each of the upstream and downstream primers of the two pairs of primers, Taq enzyme 1ul, DNA template 2ul, ultrapure water 12ul; The reaction program of PCR amplification was as follows: pre-denaturation at 94°C for 3 minutes; then denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 45 seconds, for a total of 35 cycles; and finally extension at 72°C for 10 minutes and storage at 4°C.
[0025] S3. Perform electrophoresis separation on a 10% polyacrylamide gel to obtain an electrophoresis separation result.
[0026] S4. Count the genotypes of the PCR amplification products of the samples to be identified based on the electrophoresis separation results obtained in step S3 to obtain the genotype results of the samples to be identified.
[0027] S5. Cluster analysis is performed on the genotype results obtained in step S4 using the genetic analysis software MEGA to obtain the results of kinship identification of big-eyed mandarin fish: the sample individuals clustered in the same branch have a close relationship, while the sample individuals clustered in different branches have a distant relationship.
[0028] The following examples 1 and 2 further illustrate the role and advantages of the microsatellite primers and identification method of the present invention in identifying the relationship of big-eyed mandarin fish.
[0029] Example 1: Identification of the relationship between different families of bigeye mandarin fish Ten samples were taken from each of the three full-sib families of bigeye mandarin fish, and were respectively designated as samples 1-10, samples 11-20, and samples 21-30. DNA of the 30 bigeye mandarin fish samples was extracted according to the operation steps described in step S1, and PCR amplification was performed using the five sets of double PCR systems of the present invention. After the amplified products were separated by electrophoresis, the genotypes of the 30 samples at the 10 microsatellite loci were statistically analyzed, and a cluster analysis diagram of the 30 samples was drawn, as shown in the attached figure. Figure 1 shown.
[0030] Analysis Attachment Figure 1 , we get: samples 1-10 converge into one branch, samples 11-20 converge into one branch, and samples 21-30 converge into one branch.
[0031] Individuals of the same family gathered into the same branch, and individuals of different families gathered into different branches. From this result, it can be known that the microsatellite primers and identification method for identifying the kinship of big-eyed mandarin fish of the present invention can be used to identify the kinship of big-eyed mandarin fish with high accuracy.
[0032] Example 2: Identification of kinship of random bigeye mandarin fish samples Twenty bigeye mandarin fish samples were collected from the wild, and the method of the present invention was used to identify the relationship of the 20 random samples: DNA of the 20 samples was extracted according to the operation of step S1, and then the extracted DNA of the 20 samples was amplified by PCR using the microsatellite primers of the present invention. After the amplified products were separated by electrophoresis on a 10% polyacrylamide gel, the genotypes of the 20 samples in 5 groups of double PCR systems were counted, and a cluster analysis diagram of the 20 samples was drawn. The cluster analysis diagram is shown in the attached figure. Figure 2 shown.
[0033] Analysis Attachment Figure 2 , and we get: samples 3, 17, 13, 16, and 2 converge into one branch, samples 20, 1, and 12 converge into one branch, samples 15 and 18 converge into one branch, and samples 6, 9, 19, 5, 10, 14, 8, 7, 4, and 11 converge into one branch.
[0034] The individuals gathered in the same branch are closely related, and those from different branches can mate and reproduce. Before breeding big-eyed mandarin fish, their relationship should be identified first to avoid inbreeding.
[0035] This example further shows that the primers and identification method for identifying the kinship of big-eyed mandarin fish of the present invention can guide the artificial breeding of big-eyed mandarin fish farms and avoid the phenomenon of inbreeding.
Claims
1. A microsatellite primer for identifying the genetic relationship of big-eyed mandarin fish, characterized in that: The microsatellite primers include: nucleotide sequences shown in SEQ ID NO.1-20.
2. A microsatellite primer for identifying the genetic relationship of bigeye mandarin fish according to claim 1, characterized in that: The nucleotide sequences shown in SEQ ID NO.1-20 constitute 10 pairs of microsatellite primer sets; The 10 pairs of microsatellite primers constitute 5 sets of duplex PCR systems; The information of the 5 sets of dual PCR systems is: 。 3. A method for identifying the kinship of big-eyed mandarin fish, characterized in that: The identification method comprises the following steps: S1, extracting DNA from samples to be identified; S2. Performing PCR amplification on the sample DNA extracted in step S1 using the microsatellite primers described in claim 1 or 2 to obtain an amplified product; S3, performing electrophoresis separation on a 10% polyacrylamide gel on the amplified product obtained in step S2 to obtain an electrophoresis separation result; S4, counting the genotypes of the PCR amplification products of the samples to be identified according to the electrophoresis separation results obtained in step S3, to obtain the genotype results of the samples to be identified; S5. Perform cluster analysis on the genotype results obtained in step S4 by using genetic analysis software to obtain the results of kinship identification of big-eyed mandarin fish: the sample individuals gathered in the same branch have a close relationship, while the sample individuals gathered in different branches have a distant relationship.
4. A method for identifying the kinship of bigeye mandarin fish according to claim 3, characterized in that: In step S2, the reaction system for PCR amplification is 25ul: 3ul of 10×PCR Buffer, 1ul of 2.5mmol / L dNTP, 3ul of MgCl2, 1ul of each of the upstream and downstream primers of two pairs of primers, 1ul of Taq enzyme, 2ul of DNA template, and 12ul of ultrapure water.
5. The method for identifying the kinship of bigeye mandarin fish according to claim 3, characterized in that: In step S2, the reaction procedure of PCR amplification is: pre-denaturation at 94°C for 3 minutes; then denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 45 seconds, for a total of 35 cycles; and finally extension at 72°C for 10 minutes and storage at 4°C.
6. A method for identifying the kinship of bigeye mandarin fish according to claim 3, characterized in that: In step S5, the genetic analysis software used is MEGA software.