SSR markers, amplification primers and their applications for tracking released populations of full-sib families of leopard gill bass

Abstract

The present invention provides an SSR marker for tracking a released population of full-sib family leopard gill perch, the SSR marker comprising a nucleotide sequence such as PLHB-01 shown in SEQ ID NO.1, a nucleotide sequence such as PLHB-02 shown in SEQ ID NO.2, a nucleotide sequence such as PLHB-03 shown in SEQ ID NO.3, a nucleotide sequence such as PLHB-04 shown in SEQ ID NO.4, a nucleotide sequence such as PLHB-05 shown in SEQ ID NO.5, a nucleotide sequence such as PLHB-06 shown in SEQ ID NO.6, a nucleotide sequence such as PLHB-07 shown in SEQ ID NO.7, and a nucleotide sequence such as PLHB-08 shown in SEQ ID NO.8. The present invention also provides amplification primers and uses of the SSR marker, and a method for tracking a released population of full-sib family leopard gill perch. The SSR molecular marker developed by the present invention can be used for identification of artificially released leopard gill perch, and is widely used in artificial proliferation and release of leopard gill perch.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and particularly relates to an SSR marker for tracking a released population of full-sib family leopard gill perch, an amplification primer and an application thereof. Background Art

[0002] Leopard gill perch (Plectropomus Leopardus) belongs to the Perciformes order, Perciformes suborder, Serranidae family, and the genus Plectropomus. It is a warm-water island reef marine fish, mainly distributed in the waters from the western Pacific Ocean to the Indian Ocean. It is an important farmed fish in southern my country. Leopard gill perch has tender meat and delicious taste. It has the characteristics of low fat, low cholesterol and high protein. It is a high-end edible fish, deeply loved by consumers and has broad market prospects. However, the natural number of leopard gill perch is scarce, and the high intensity of fishing pressure in recent years has caused its resources to be exhausted. In order to solve the problem of the sharp decline in the biomass of leopard gill perch in my country's natural waters, artificially propagated leopard gill perch seedlings are usually released into the release sea area to restore or increase the natural population.

[0003] Artificially propagated and released fish fry may not be able to adapt and survive in natural waters. The recapture rate refers to the percentage of the number of released aquatic animals recaptured after a certain period of time to the total number of released individuals. The key to calculating the recapture rate of propagated and released fish fry is to identify the released population from the recaptured population, and then analyze the survival and spread of the released population in the natural environment, as well as the impact on the local wild population. Commonly used marks for identifying released populations include hanging tags, fin clipping, branding and fluorescent pigments, which have disadvantages such as low mark retention rate and affecting the behavior and growth of marked fish.

[0004] DNA molecular markers are a new type of marker technology that detects genetic differences at the DNA level. They not only overcome the above-mentioned marker shortcomings, but also have high polymorphism and more efficient population identification. SSR (Simple Sequence Repeats) markers are a molecular marker technology based on specific primer PCR developed in recent years. They are also called microsatellite DNA, which is a type of tandem repeat sequence of dozens of nucleotides consisting of several nucleotides (generally 1 to 6) as repeating units. Microsatellites are randomly distributed on chromosomes, and their polymorphism is caused by different numbers of repetitions and incomplete repetitions, resulting in a high level of allele polymorphism.

[0005] Artificial insemination is generally used for artificial propagation and release of leopard gill perch fry, and the parents are clear. The same batch of released fry are generally full-sib families with a clear genetic background, and each SSR locus has a maximum of 4 alleles. Analysis through SSR marker molecular marker technology can accurately, simply and quickly identify the released fry. Summary of the invention

[0006] The purpose of the present invention is to provide an SSR marker for tracking a released population of full-sib family leopard gill perch in response to the above technical problems to be solved. The marker can effectively identify and distinguish whether the leopard gill perch recaptured in the wild is an artificially released population.

[0007] To achieve the above objectives, the present invention provides an SSR marker for tracking a released population of full-sib family leopard gill bass, the SSR marker comprising PLHB-01 as shown in SEQ ID NO.1, PLHB-02 as shown in SEQ ID NO.2, PLHB-03 as shown in SEQ ID NO.3, PLHB-04 as shown in SEQ ID NO.4, PLHB-05 as shown in SEQ ID NO.5, PLHB-06 as shown in SEQ ID NO.6, PLHB-07 as shown in SEQ ID NO.7, and PLHB-08 as shown in SEQ ID NO.8.

[0008] On the other hand, the present invention also provides an amplification primer for tracking SSR markers of a released population of full-sib family leopard gill perch, the amplification primer comprising:

[0009] PLHB-01-F: 5'-AAAAGGTCAGGCCAGCTCTC-3';

[0010] PLHB-01-R: 5'-TGCTGCTGGTAATGCACTGA-3';

[0011] PLHB-02-F: 5'-TGACGGTCATGTCTTTGAGGG-3';

[0012] PLHB-02-R: 5'-CAACAGTACCTCCCAGCGAG-3';

[0013] PLHB-03-F: 5'-CTCTTTGTCCTCAGACCCCC-3';

[0014] PLHB-03-R: 5'-TCAAAGGAGCACCCAAGTCA-3';

[0015] PLHB-04-F: 5'-AGTCGTTTTTCGTGGCGTTG-3';

[0016] PLHB-04-R: 5'-GTCGACCACTCCGAAACAT-3';

[0017] PLHB-05-F: 5'-GGTAGCTCACCTGCCAAAGT-3';

[0018] PLHB-05-R: 5'-TGCACCGAGATGCTGTTTGA-3';

[0019] PLHB-06-F: 5'-AGCTTTGTTGCAGCCTCTGA-3';

[0020] PLHB-06-R: 5'-CAGCAGCATGAACAGGCAAG-3';

[0021] PLHB-07-F: 5'-CAGTGCGCTGATTTGAGGCTG-3';

[0022] PLHB-07-R: 5'-AACTGAGTAGCAGGTGGCAC-3';

[0023] PLHB-08-F: 5'-AGGTGTTTACAGACGTAAGGT-3';

[0024] PLHB-08-R: 5'-TGACAACTGTGTTGCACTCT-3'.

[0025] On the other hand, the present invention also provides a method for detecting the SSR markers used to track the released population of full-sib family leopard gill perch, using the genomic DNA of the released population sample of leopard gill perch as a template and using the amplification primers to perform PCR amplification.

[0026] On the other hand, the present invention also provides the use of the SSR marker for tracking a released population of full-sib family leopard gill perch for tracking a released population of full-sib family leopard gill perch.

[0027] On the other hand, the present invention also provides a method for typing SSR markers of a released population of full-sib family leopard gill perch, comprising the following steps:

[0028] (1) Collect at least 10 full-sibling leopard gill perch from artificially released and recaptured individuals, sample the dorsal fin or caudal fin, and extract genomic DNA from the leopard gill perch;

[0029] (2) Using the extracted genomic DNA as a template, PCR amplification was performed using primer pairs PLHB-01-F and PLHB-01-R for the PLHB-01 site, primer pairs PLHB-02-F and PLHB-02-R for the PLHB-02 site, primer pairs PLHB-03-F and PLHB-03-R for the PLHB-03 site, primer pairs PLHB-04-F and PLHB-04-R for the PLHB-04 site, primer pairs PLHB-05-F and PLHB-05-R for the PLHB-05 site, primer pairs PLHB-06-F and PLHB-06-R for the PLHB-06 site, primer pairs PLHB-07-F and PLHB-07-R for the PLHB-07 site, and primer pairs PLHB-08-F and PLHB-08-R for the PLHB-08 site;

[0030] (3) performing capillary electrophoresis on the PCR amplification product to obtain an electrophoresis peak diagram;

[0031] (4) Interpret the obtained electrophoresis peak diagram to obtain the typing result.

[0032] Preferably, in step (2), the reaction system for PCR amplification is: Mg-free 2+ 2.5 μL of 10× PCR buffer, 2.0 μL of 25 mM MgCl2 aqueous solution, 0.5 μL of 10 mM dNTP, 1 U of high-fidelity PCR enzyme, 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM reverse primer, 12.5 ng of DNA template, and make up to 25 μL with sterile double distilled water.

[0033] Preferably, in step (2), the reaction procedure of the PCR amplification is: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 30 seconds, for a total of 35 cycles; and extension at 72°C for another 6 minutes.

[0034] Preferably, in step (3), the 5' end of the amplification primer is labeled with a FAM fluorescent group, and the genotyping sequencer ABI 3730XL is used for capillary electrophoresis typing.

[0035] Preferably, in step (4), peak graph interpretation is performed using GeneMarker4.0.

[0036] On the other hand, the present invention also provides a method for tracking a released population of full-sib family leopard gill perch, comprising the following steps:

[0037] (1) Collect at least 10 full-sibling leopard gill perch from artificially released and recaptured individuals, sample the dorsal fin or caudal fin, and extract genomic DNA from the leopard gill perch;

[0038] (2) Using the extracted genomic DNA as a template, PCR amplification was performed using primer pairs PLHB-01-F and PLHB-01-R for the PLHB-01 site, primer pairs PLHB-02-F and PLHB-02-R for the PLHB-02 site, primer pairs PLHB-03-F and PLHB-03-R for the PLHB-03 site, primer pairs PLHB-04-F and PLHB-04-R for the PLHB-04 site, primer pairs PLHB-05-F and PLHB-05-R for the PLHB-05 site, primer pairs PLHB-06-F and PLHB-06-R for the PLHB-06 site, primer pairs PLHB-07-F and PLHB-07-R for the PLHB-07 site, and primer pairs PLHB-08-F and PLHB-08-R for the PLHB-08 site;

[0039] (3) performing capillary electrophoresis on the PCR amplification product to obtain an electrophoresis peak diagram;

[0040] (4) Interpreting the obtained electrophoresis peak diagram to obtain typing results;

[0041] (5) Establishing an allele matrix using the alleles of the recaptured individuals of the leopard gill perch to be tested, as well as the allele typing results of the released population and the wild population;

[0042] (6) calculating the genetic distance between the samples for the alleles of the captured individuals of the tested leopard gill perch, as well as the alleles of the released population and the wild population;

[0043] (7) Based on the obtained genetic distance, cluster analysis is performed to construct a cluster tree, and the cluster tree results are used to determine whether the recaptured individuals of the leopard gill perch to be tested are from the released population.

[0044] Preferably, in step (2), the reaction system for PCR amplification is: Mg-free 2+ 2.5 μL of 10× PCR buffer, 2.0 μL of 25 mM MgCl2 aqueous solution, 0.5 μL of 10 mM dNTP, 1 U of high-fidelity PCR enzyme, 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM reverse primer, 12.5 ng of DNA template, and make up to 25 μL with sterile double distilled water.

[0045] Preferably, in step (2), the reaction procedure of the PCR amplification is: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 30 seconds, for a total of 35 cycles; and extension at 72°C for another 6 minutes.

[0046] Preferably, in step (3), the 5' end of the amplification primer is labeled with a FAM fluorescent group, and the genotyping sequencer ABI 3730XL is used for capillary electrophoresis typing.

[0047] Preferably, GeneMarker4.0 is used for peak plot interpretation and allele matrix construction.

[0048] Preferably, genetic distance calculations are performed using GenALEx.

[0049] Preferably, MEGA6.0 is used to construct the cluster tree according to the UPGMA method.

[0050] The SSR molecular marker developed by the invention can be used for identification of artificially released leopard gill perch, and is widely used in artificial propagation and release of leopard gill perch. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is the identification result of the artificially released group of 38 leopard gill bass recaptured in August 2021.

[0052] Figure 2 This is the identification result of the first and second artificially released groups of 20 leopard spiny bass recaptured in February 2022. DETAILED DESCRIPTION

[0053] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0054] It is worth noting that the experimental methods in the following examples, unless otherwise specified, are all conventional methods or carried out according to the instructions of the kit. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0055] Primer synthesis and sequencing were completed by Wuhan Tianyi Huiyuan Co., Ltd.

[0056] As used herein, a "full-sib family" refers to a collection of children born to the same parents.

[0057] The genome of the spiny gill perch was downloaded from Genbank, with the submission number PRJDB9369, a total length of 748.23Mb, a GC content of 39.5%, a scaffold N50 length of 30.01Mb, and a contig N50 length of 1.08Mb. The SSR sequence was predicted using the misa software, SSRs with more than 4 repeat units were selected, and the SSR loci with the highest expected heterozygosity were calculated. Primer3 software was used to batch design primers with a PCR amplification product length of 200 to 300bp for the screened SSRs. The primers were filtered, and the free energy was required to be greater than 30 (no hairpin structure). Using the filtered primers, a genome Blast alignment was performed to select specific primers. Theoretically, no sequence was amplified in the non-SSR region, and it could be amplified in the SSR region. The SSR amplified fragments were selected to have high polymorphism, good and stable amplification, high heterozygosity, different amplified fragment sizes, the same or similar annealing temperature (58-60°C), and the GC value was controlled between 50% and 60%.

[0058] After screening, the 8 SSR markers obtained all have 4 repeating units, which are more accurate in identifying the released population. The 8 SSR markers obtained are PLHB-01, PLHB-02, PLHB-03, PLHB-04, PLHB-05, PLHB-06PLHB-07, and PLHB-08, and their nucleotide sequences are shown in SEQ ID NO.1 to 8, respectively.

[0059] In order to achieve PCR amplification of the above 8 SSR markers, the specific amplification primers were designed as follows:

[0060] (1) Targeting the PLHB-01 site:

[0061] PLHB-01-F: 5'-AAAAGGTCAGGCCAGCTCTC-3' (SEQ ID NO.9);

[0062] PLHB-01-R: 5'-TGCTGCTGGTAATGCACTGA-3' (SEQ ID NO. 10);

[0063] (2) Targeting the PLHB-02 site:

[0064] PLHB-02-F: 5'-TGACGGTCATGTCTTTGAGGG-3' (SEQ ID NO. 11);

[0065] PLHB-02-R: 5'-CAACAGTACCTCCCAGCGAG-3' (SEQ ID NO. 12);

[0066] (3) Targeting the PLHB-03 site:

[0067] PLHB-03-F: 5'-CTCTTTGTCCTCAGACCCCC-3' (SEQ ID NO. 13);

[0068] PLHB-03-R: 5'-TCAAAGGAGCACCCAAGTCA-3' (SEQ ID NO. 14);

[0069] (4) Targeting the PLHB-04 site:

[0070] PLHB-04-F: 5'-AGTCGTTTTTCGTGGCGTTG-3' (SEQ ID NO. 15);

[0071] PLHB-04-R: 5'-GTCGACCACTCCGAAACAT-3' (SEQ ID NO. 16);

[0072] (5) Targeting the PLHB-05 site:

[0073] PLHB-05-F: 5'-GGTAGCTCACCTGCCAAAGT-3' (SEQ ID NO. 17);

[0074] PLHB-05-R: 5'-TGCACCGAGATGCTGTTTGA-3' (SEQ ID NO. 18);

[0075] (6) Targeting the PLHB-06 site:

[0076] PLHB-06-F: 5'-AGCTTTGTTGCAGCCTCTGA-3' (SEQ ID NO. 19);

[0077] PLHB-06-R: 5'-CAGCAGCATGAACAGGCAAG-3' (SEQ ID NO. 20);

[0078] (7) Targeting the PLHB-07 site:

[0079] PLHB-07-F: 5'-CAGTGCGCTGATTTGAGGCTG-3' (SEQ ID NO. 21);

[0080] PLHB-07-R: 5'-AACTGAGTAGCAGGTGGCAC-3' (SEQ ID NO. 22);

[0081] (8) Targeting PLHB-08 site:

[0082] PLHB-08-F: 5'-AGGTGTTTACAGACGTAAGGT-3' (SEQ ID NO. 23);

[0083] PLHB-08-R: 5'-TGACAACTGTGTTGCACTCT-3' (SEQ ID NO. 24).

[0084] The SSR markers of the released population of full-sib pedigree leopard gill perch were typed as follows:

[0085] (1) Collect at least 10 full-sibling leopard gill perch from artificial release and recapture, and sample the dorsal fin or caudal fin. Use the Marine Animal Tissue Genomic DNA Extraction Kit (Tiangen Company, Cat. No.: DP324-02) to extract the genomic DNA of the leopard gill perch.

[0086] (2) Using the extracted genomic DNA as a template, PCR amplification was performed using the primer pairs PLHB-01-F and PLHB-01-R for the PLHB-01 site, the primer pairs PLHB-02-F and PLHB-02-R for the PLHB-02 site, the primer pairs PLHB-03-F and PLHB-03-R for the PLHB-03 site, the primer pairs PLHB-04-F and PLHB-04-R for the PLHB-04 site, the primer pairs PLHB-05-F and PLHB-05-R for the PLHB-05 site, the primer pairs PLHB-06-F and PLHB-06-R for the PLHB-06 site, the primer pairs PLHB-07-F and PLHB-07-R for the PLHB-07 site, and the primer pairs PLHB-08-F and PLHB-08-R for the PLHB-08 site. PCR used Takara's Ex Taq enzyme system (Cat. No.: RR001A), and the amplification reaction system was: Mg-free 2+ 2.5 μL of 10×PCR buffer, 2.0 μL of 25 mM MgCl2 aqueous solution, 0.5 μL of 10 mM dNTP, 0.05 μL of high-fidelity PCR enzyme, 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM reverse primer, 12.5 ng of DNA template, and add sterile double distilled water to 25 μL. The reaction program of PCR amplification is: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 30 seconds, for a total of 35 cycles; and extension at 72°C for another 6 minutes. Each PCR reaction has a pair of primers, and 16 reactions are performed on one DNA sample.

[0087] (3) Perform capillary electrophoresis on the PCR amplification product to obtain an electrophoresis peak diagram. The 5' end of the amplification primer can be labeled with a FAM fluorescent group, and the genotyping sequencer ABI 3730XL can be used for capillary electrophoresis typing.

[0088] (4) The obtained electrophoresis peak pattern was interpreted using the software GeneMarker4.0 (Softgenetics) to obtain the typing result.

[0089] The specific steps of the method for tracking the released population of full-sib family leopard gill bass are as follows:

[0090] (1) Collect at least 10 full-sibling leopard gill perch from artificial release and recapture, and sample the dorsal fin or caudal fin. Use the Marine Animal Tissue Genomic DNA Extraction Kit (Tiangen Company, Cat. No.: DP324-02) to extract the genomic DNA of the leopard gill perch.

[0091] (2) Using the extracted genomic DNA as a template, PCR amplification was performed using the primer pairs PLHB-01-F and PLHB-01-R for the PLHB-01 site, the primer pairs PLHB-02-F and PLHB-02-R for the PLHB-02 site, the primer pairs PLHB-03-F and PLHB-03-R for the PLHB-03 site, the primer pairs PLHB-04-F and PLHB-04-R for the PLHB-04 site, the primer pairs PLHB-05-F and PLHB-05-R for the PLHB-05 site, the primer pairs PLHB-06-F and PLHB-06-R for the PLHB-06 site, the primer pairs PLHB-07-F and PLHB-07-R for the PLHB-07 site, and the primer pairs PLHB-08-F and PLHB-08-R for the PLHB-08 site. PCR used Takara's Ex Taq enzyme system (Cat. No.: RR001A), and the amplification reaction system was: Mg-free 2+ 2.5 μL of 10×PCR buffer, 2.0 μL of 25 mM MgCl2 aqueous solution, 0.5 μL of 10 mM dNTP, 0.05 μL of high-fidelity PCR enzyme, 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM reverse primer, 12.5 ng of DNA template, and add sterile double distilled water to 25 μL. The reaction program of PCR amplification is: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 30 seconds, for a total of 35 cycles; and extension at 72°C for another 6 minutes. Each PCR reaction has a pair of primers, and 16 reactions are performed on one DNA sample.

[0092] (3) Perform capillary electrophoresis on the PCR amplification product to obtain an electrophoresis peak diagram.

[0093] (4) The obtained electrophoresis peak pattern was interpreted using the software GeneMarker4.0 (Softgenetics) to obtain the typing result.

[0094] (5) The allele matrix was established using the software GeneMarker4.0 (Softgenetics) using the alleles of the recaptured individuals of the tested leopard gill perch and the allele typing results of the released population and the wild population.

[0095] (6) The genetic distance between the samples was calculated using the software GenALEx for the alleles of the captured individuals of the tested leopard gill perch, as well as the alleles of the released population and the wild population.

[0096] (7) Based on the obtained genetic distance, cluster analysis was performed, and a cluster tree was constructed using the software MEGA6.0 according to the UPGMA method (unweighted pair-group method using an arithmetic average). The cluster tree results were used to determine whether the recaptured individuals of the leopard gill perch to be tested were from the released population.

[0097] Example 1

[0098] In April 2021, a batch of full-sib leopard gill perch were artificially released in the waters of Beihai, Guangxi, and 20 samples were collected (FL). In August 2021, 38 leopard gill perch were recaptured in the waters of Beihai, Guangxi (HB). A total of 58 leopard gill perch samples were extracted, and PCR amplification and capillary electrophoresis typing were performed using the above 8 pairs of SSR marker amplification primers according to the above amplification reaction system and conditions. GeneMarker4.0 was used for peak interpretation, and the samples were combined with 36 known wild leopard gill perch samples caught in the Beihai waters of Guangxi (YS) for analysis and construction of the allele matrix. GenALEx was used for genetic distance calculation, and MEGA6.0 was used to construct a cluster tree using the UPGMA method.

[0099] like Figure 1 As shown in the figure, the clustering results show that among the 38 leopard gill perch recaptured in August 2021, 18 of them were clustered with the leopard gill perch artificially released in April 2021, forming an artificially released group; the other 20 were clustered with the wild leopard gill perch previously caught in the Beihai waters of Guangxi, forming a wild group. Therefore, the artificial release of leopard gill perch in April 2021 increased the natural resources of leopard gill perch in the Beihai waters by about 90% in a four-month period.

[0100] Example 2

[0101] In April 2021, a batch of full-sib leopard gill perch was artificially released in the waters of Weizhou Island, Guangxi, and 20 samples were taken (FL-1); in October 2021, a batch of full-sib leopard gill perch was artificially released in the waters of Weizhou Island, Guangxi, and 20 samples were taken (FL-2). In February 2022, 20 leopard gill perch were recaptured in the waters of Weizhou Island, Guangxi (HB). A total of 60 leopard gill perch samples were extracted, and PCR amplification and capillary electrophoresis typing were performed using the above 8 pairs of SSR marker amplification primers according to the above amplification reaction system and conditions. GeneMarker4.0 was used for peak interpretation, and the samples were combined with 36 known wild leopard gill perch samples caught in the Beihai waters of Guangxi (YS) for analysis and construction of an allele matrix. GenALEx was used to calculate genetic distances, and MEGA6.0 was used to construct a cluster tree using the UPGMA method.

[0102] like Figure 2 As shown in the figure, the clustering results show that among the 20 leopard gill perch recaptured in February 2022, 5 of them were clustered with the leopard gill perch artificially released in April 2021, forming the first artificial release group; 8 of them were clustered with the leopard gill perch artificially released in October 2021, forming the second artificial release group; and the other 7 were clustered with the wild leopard gill perch previously caught in the Beihai waters of Guangxi, forming a wild group. Therefore, among the leopard gill perch recaptured in February 2022, the proportions of the first release, second release and wild groups were 25%, 40% and 35% respectively.

[0103] The above embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements fall within the scope of protection of the present invention.

Claims

1. A SSR marker for tracking a released population of full-sib family leopard gill perch, characterized in that: The SSR marker consists of PLHB-01 as shown in SEQ ID NO.1, PLHB-02 as shown in SEQ ID NO.2, PLHB-03 as shown in SEQ ID NO.3, PLHB-04 as shown in SEQ ID NO.4, PLHB-05 as shown in SEQ ID NO.5, PLHB-06 as shown in SEQ ID NO.6, PLHB-07 as shown in SEQ ID NO.7, and PLHB-08 as shown in SEQ ID NO.

8.

2. An amplification primer for tracking SSR markers of a released population of full-sib family leopard gill perch, the amplification primer comprising: PLHB-01-F: 5'-AAAAGGTCAGGCCAGCTCTC-3'; PLHB-01-R: 5'-TGCTGCTGGTAATGCACTGA-3'; PLHB-02-F: 5'-TGACGGTCATGTCTTTGAGGG-3'; PLHB-02-R: 5'-CAACAGTACCTCCCAGCGAG-3'; PLHB-03-F: 5'-CTCTTTGTCCTCAGACCCCC-3'; PLHB-03-R: 5'-TCAAAGGAGCACCCAAGTCA-3'; PLHB-04-F: 5'-AGTCGTTTTTCGTGGCGTTG-3'; PLHB-04-R: 5'-GTCGACCACTCCGAAACAT-3'; PLHB-05-F: 5'-GGTAGCTCACCTGCCAAAGT-3'; PLHB-05-R: 5'-TGCACCGAGATGCTGTTTGA-3'; PLHB-06-F: 5'-AGCTTTGTTGCAGCCTCTGA-3'; PLHB-06-R: 5'-CAGCAGCATGAACAGGCAAG-3'; PLHB-07-F: 5'-CAGTGCGCTGATTTGAGGCTG-3'; PLHB-07-R: 5'-AACTGAGTAGCAGGTGGCAC-3'; PLHB-08-F: 5'-AGGTGTTTACAGACGTAAGGT-3'; PLHB-08-R: 5'-TGACAACTGTGTTGCACTCT-3'.

3. A method for detecting the SSR marker according to claim 1, characterized in that: The method comprises: using the genomic DNA of the released population sample of leopard gill perch as a template and using the amplification primers described in claim 2 to perform PCR amplification.

4. Use of the amplification primers described in claim 2 for tracking a released population of full-sib family leopard gill perch.

5. A method for typing SSR markers of a released population of full-sib family leopard gill perch, comprising the following steps: (1) Collect at least 10 full-sibling leopard gill perch from artificially released and recaptured individuals, sample the dorsal fin or caudal fin, and extract the genomic DNA of the leopard gill perch; (2) using the extracted genomic DNA as a template, and using the amplification primers described in claim 2 to perform PCR amplification on the SSR marker described in claim 1; (3) Perform capillary electrophoresis on the PCR amplification product to obtain an electrophoresis peak diagram; (4) Interpret the obtained electrophoresis peak pattern to obtain the typing result.

6. The method according to claim 5, characterized in that In step (2), the reaction system for PCR amplification is: Mg-free 2+ 2.5 μL of 10× PCR buffer, 2.0 μL of 25 mM MgCl2 aqueous solution, 0.5 μL of 10mM dNTP, 1 U of high-fidelity PCR enzyme, 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM reverse primer, 12.5 ng of DNA template, and make up to 25 μL with sterile double distilled water.

7. The method according to claim 5, characterized in that In step (2), the reaction procedure of the PCR amplification is: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 30 seconds, for a total of 35 cycles; and extension at 72°C for another 6 minutes.

8. A method for tracking a released population of full-sib family leopard gill perch, comprising the following steps: (1) Collect at least 10 full-sibling leopard gill perch from artificially released and recaptured individuals, sample the dorsal fin or caudal fin, and extract the genomic DNA of the leopard gill perch; (2) using the extracted genomic DNA as a template, and using the amplification primers described in claim 2 to perform PCR amplification on the SSR marker described in claim 1; (3) Perform capillary electrophoresis on the PCR amplification product to obtain an electrophoresis peak diagram; (4) Interpret the obtained electrophoresis peak diagram to obtain the typing result; (5) Establish an allele matrix using the alleles of the recaptured individuals of the leopard perch to be tested, as well as the allele typing results of the released population and the wild population; (6) Calculate the genetic distance between the alleles of the captured individuals of the tested leopard gill perch, as well as the alleles of the released population and the wild population; (7) Based on the obtained genetic distance, cluster analysis was performed to construct a cluster tree. The cluster tree results were used to determine whether the recaptured individuals of the tested leopard gill perch were from the released population.

9. The method according to claim 8, characterized in that In step (2), the reaction system for PCR amplification is: Mg-free 2+ 2.5 μL of 10× PCR buffer, 2.0 μL of 25 mM MgCl2 aqueous solution, 0.5 μL of 10mM dNTP, 1 U of high-fidelity PCR enzyme, 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM reverse primer, 12.5 ng of DNA template, and make up to 25 μL with sterile double distilled water.

10. The method according to claim 8, characterized in that: In step (2), the reaction procedure of the PCR amplification is: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 30 seconds, for a total of 35 cycles; and extension at 72°C for another 6 minutes.

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