SSR molecular marker primer set for identifying original species of ptereleotris rubripectoralis in weizhou island of guangxi and application thereof

Abstract

The application provides a kind of to identify the SSR molecular marker primer group of Guangxi Weizhou Island leopard pattern branchlet puffer original species and application thereof.The application obtains a group of SSR molecular markers capable of identifying Guangxi Weizhou Island leopard pattern branchlet puffer original species: PLWZ-01, PLWZ-02, PLWZ-03, PLWZ-04, PLWZ-05, PLWZ-06, the nucleotide sequences of which are shown in SEQ ID NO.1-6, the SSR molecular markers have the characteristics of high polymorphism but low polymorphism in the original population of Guangxi Weizhou Island;the primer group for amplifying the SSR molecular markers is obtained by screening, and has stable amplification and good repeatability;at the same time, a method for identifying the original species of Guangxi Weizhou Island leopard pattern branchlet puffer is established, which solves the problem that the existing leopard pattern branchlet puffer geographical population is not clear and the original species parent of the seedling of propagation and release is difficult to identify.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of molecular biology technology. More particularly, it relates to a set of SSR molecular marker primers for identifying Plectropomus leopardus in Weizhou Island, Guangxi, and application thereof. BACKGROUND

[0002] Plectropomus leopardus, commonly known as "eastern star", belongs to Perciformes, Perciformes, Serranidae, Epinephelinae and Plectropomus, and is a warm water island reef fish mainly distributed in the western Pacific Ocean to the Indian Ocean. It is an important farmed fish in southern China. Plectropomus leopardus has tender and delicious meat, low fat, low cholesterol and high protein, and is a high-grade edible fish, which is deeply loved by consumers and has a broad market prospect.

[0003] Weizhou Island is located in the central Beibu Gulf of Beihai City, Guangxi Zhuang Autonomous Region. It is north of Beihai City, east of Leizhou Peninsula, adjacent to Xieyang Island in the southeast, south of Hainan Island across the sea, and west of Vietnam. Weizhou Island is located in the northern margin of the tropical coral distribution, and its geographical specificity makes it have very high ecological value. Fishes in Weizhou Island coral reef sea are mainly Perciformes, which accounts for 74.56% of the total number of collected species. Plectropomus leopardus is one of the characteristic fishes in Weizhou Island. However, with the promotion of the construction of Guangxi Beibu Gulf Economic Zone and the intensification of human activities such as tourism resources and marine resources development, the resources of Plectropomus leopardus in this sea area have decreased sharply.

[0004] It is an effective method to restore or increase the natural population by releasing artificially propagated juvenile Ptereleotris lunaris into Weizhou Island waters. However, Ptereleotris lunaris from different sea areas will have differences in color, taste, and appearance. Releasing Ptereleotris lunaris bred from parents from other sea areas into Weizhou Island waters will pollute the germplasm resources of the sea area. Ptereleotris lunaris parents are very difficult to obtain, and it is impossible to obtain enough parents for breeding and releasing through field capture. At the same time, it is very difficult to determine the original place of Ptereleotris lunaris parents by naked eye. At present, there are studies on the genetic structure and genetic variation of Ptereleotris lunaris population using microsatellite molecular markers. For example, the prior art discloses a method for constructing Ptereleotris lunaris microsatellite DNA molecular markers. By designing specific primers for microsatellite markers, Ptereleotris lunaris microsatellite markers with high polymorphism are developed and applied to the study of Ptereleotris lunaris population genetic structure and genetic breeding. However, the method only provides and verifies 7 primers, and 14649 sites can be detected in Ptereleotris lunaris. If used for different population genetic structure analysis and verification, thousands of specific primers need to be designed, which is complex, time-consuming and laborious. At the same time, the microsatellite marker primers designed by the method cannot identify and distinguish the original species of Ptereleotris lunaris. Therefore, it is necessary to establish a reliable molecular biology technology for identifying the original species of Ptereleotris lunaris in Weizhou Island, Guangxi. SUMMARY

[0005] The purpose of the present application is to provide a set of SSR molecular marker primers for identifying the original species of Ptereleotris lunaris in Weizhou Island, Guangxi, and its application, to solve the problem that the existing Ptereleotris lunaris geographic population is not clearly identified, and the original species of the parent of the released and released seedling is difficult to distinguish.

[0006] The first purpose of the present application is to provide a set of SSR molecular markers for identifying the original species of Ptereleotris lunaris in Weizhou Island, Guangxi.

[0007] The second purpose of the present application is to provide a primer set for amplifying the SSR molecular markers.

[0008] The third purpose of the present application is to provide the application of the SSR molecular markers or the primer set.

[0009] The fourth purpose of the present application is to provide a detection kit for identifying the original species of Ptereleotris lunaris in Weizhou Island, Guangxi.

[0010] The fifth purpose of the present application is to provide a method for identifying the original species of Ptereleotris lunaris in Weizhou Island, Guangxi.

[0011] The sixth purpose of the present application is to provide the application of the method for identifying the original species of Ptereleotris lunaris in Weizhou Island, Guangxi.

[0012] The above purposes of the present application are achieved by the following technical solutions:

[0013] The application obtains SSR molecular markers by designing the genome of Plectryagus leucopterus (submission number PRJDB9369, total length 748.23 Mb, GC content 39.5%, scaffold N50 length 30.01 Mb, contig N50 length 1.08 Mb.), and the SSR molecular markers are PLWZ-01, PLWZ-02, PLWZ-03, PLWZ-04, PLWZ-05, PLWZ-06, the nucleotide sequences of which are shown in SEQ ID NO. 1-6 respectively. It is shown by verification of the above-mentioned SSR molecular markers that the six SSR sites provided by the application have the characteristics of high polymorphism but low polymorphism in the Weizhou Island original population in Guangxi; and the primer groups capable of amplifying the above-mentioned SSR molecular markers are screened.

[0014] The primer sequences for amplifying PLWZ-01 are shown in SEQ ID NO. 7-8 respectively;

[0015] The primer sequences for amplifying PLWZ-02 are shown in SEQ ID NO. 9-10 respectively;

[0016] The primer sequences for amplifying PLWZ-03 are shown in SEQ ID NO. 11-12 respectively;

[0017] The primer sequences for amplifying PLWZ-04 are shown in SEQ ID NO. 13-14 respectively;

[0018] The primer sequences for amplifying PLWZ-05 are shown in SEQ ID NO. 15-16 respectively;

[0019] The primer sequences for amplifying PLWZ-06 are shown in SEQ ID NO. 17-18 respectively.

[0020] The research of the application shows that the above-mentioned primer groups can be used to identify the Plectryagus leucopterus original species in the Weizhou Island sea area in Guangxi, and the primer amplification is stable and has good repeatability; meanwhile, a method for identifying the Plectryagus leucopterus original species in the Weizhou Island in Guangxi is established, the specific alleles of the Plectryagus leucopterus original species in the Weizhou Island in Guangxi can be used to perform UPGMA clustering analysis and construct a clustering tree, so that whether the Plectryagus leucopterus geographical population belongs to the Plectryagus leucopterus original species in the Weizhou Island in Guangxi can be directly identified.

[0021] Therefore, the application provides the application of the SSR molecular markers or the primer groups in identifying the Plectryagus leucopterus original species in the Weizhou Island in Guangxi or preparing a detection kit for identifying the Plectryagus leucopterus original species in the Weizhou Island in Guangxi.

[0022] The application provides a detection kit for identifying the original Ptereleotris punctata in Weizhou Island, Guangxi, containing the SSR molecular marker or the primer set.

[0023] The application provides a method for identifying the original Ptereleotris punctata in Weizhou Island, Guangxi, comprising the following steps:

[0024] S1. Collecting samples of Ptereleotris punctata to be identified, and extracting DNA from the samples;

[0025] S2. Using the DNA extracted in step S1 as a template, and performing PCR amplification by using the primer set;

[0026] S3. Performing capillary electrophoresis typing on the PCR product amplified in step S2;

[0027] S4. Interpreting the peak graph and constructing an allele matrix according to the typing result obtained in step S3;

[0028] S5. Comparing the alleles of the Ptereleotris punctata to be detected obtained in step S4 with the alleles of the known original Ptereleotris punctata in Weizhou Island, Guangxi, and calculating the genetic distance between the samples;

[0029] S6. Performing cluster analysis on the genetic distance obtained in step S5, constructing a cluster tree, and determining whether the Ptereleotris punctata to be detected is the original Ptereleotris punctata in Weizhou Island through the cluster tree.

[0030] Preferably, the reaction system for PCR amplification in step S2 is as follows: 10x PCR buffer 2.5 L without Mg 2+ , 25 mM MgCl2 2.0 L, 10 mM dNTP 0.5 L, high-fidelity PCR enzyme 1 U, 10 M forward primer 0.5 L, 10 M reverse primer 0.5 L, DNA template 12.5 ng, and the rest is supplemented with sterile double distilled water to 25 L.

[0031] Preferably, the reaction program for PCR amplification in step S2 is as follows: 95℃ pre-denaturation for 5 minutes; 95℃ denaturation for 30 seconds, 60℃ annealing for 30 seconds, 72℃ extension for 30 seconds, a total of 35 cycles; and 72℃ re-extension for 6 minutes.

[0032] Preferably, the capillary electrophoresis typing in step S3 labels the 5' end of the primer set in step S2 with a FAM fluorescent group, and performs capillary electrophoresis typing by using an ABI 3730XL genotype typing sequencer.

[0033] Preferably, the alleles of the known original Ptereleotris punctata in Weizhou Island, Guangxi, in step S5 include:

[0034] The PLWZ-01 site is: 258, 261, 267;

[0035] PLWZ-02 sites are: 195, 279, 282, 288;

[0036] PLWZ-03 sites are: 238, 251, 256, 279;

[0037] PLWZ-04 sites are: 237, 252, 255, 258;

[0038] PLWZ-05 sites are: 257, 272, 291;

[0039] PLWZ-06 sites are: 178, 199, 215, 219.

[0040] The application also provides application of the method in identifying geographical populations of Plectropomus leopardus or identifying original species parents of breeding and releasing seedlings.

[0041] The application has the following beneficial effects:

[0042] The application designs a SSR molecular marker for identifying the original species of Plectropomus leopardus in Weizhou Island, Guangxi: PLWZ-01, PLWZ-02, PLWZ-03, PLWZ-04, PLWZ-05 and PLWZ-06, the nucleotide sequences of which are shown in SEQ ID NO. 1-6, the SSR molecular marker has the characteristics of high polymorphism but low polymorphism in the original population in Weizhou Island, Guangxi, and can effectively distinguish the original population of Plectropomus leopardus in Weizhou Island, Guangxi from other populations. A primer group capable of amplifying the above-mentioned SSR molecular marker is also screened, the primer group has stable amplification and good repeatability; meanwhile, a method for identifying the original species of Plectropomus leopardus in Weizhou Island, Guangxi is established, whether the Plectropomus leopardus to be identified is the original species is determined through specific alleles and a clustering tree, and the problems that the geographical populations of Plectropomus leopardus are not clearly identified and the original species parents of breeding and releasing seedlings are difficult to identify are solved. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 Fig. 1 is a genetic relationship diagram of 173 Plectropomus leopardus, wherein 36 Plectropomus leopardus from the sea area of Weizhou Island, Guangxi (GX-WZ) are included, and the gray marked area represents the branch of the original species in Weizhou Island, Guangxi, and the Plectropomus leopardus is from Weizhou Island (GX-WZ), Dongfang in Hainan (HN-DF), Sanya in Hainan (HN-SY), Wenchang in Hainan (HN-WC), Zhanjiang in Guangdong (GD-ZJ) and Malaysia (GW-Ma), Indonesia (GW-In), Philippines (GW-Ph) and Australia (GW-Au);

[0044] Figure 2To identify whether the 1 captured Ptereleotris lunifasciata in Guangxi Beihai sea area is the original species of Weizhou Island, T is the test sample, and the rest of the reference samples include: Guangxi Weizhou (GX-WZ), Hainan Dongfang (HN-DF), Hainan Sanya (HN-SY), Hainan Wenchang (HN-WC), Guangdong Zhanjiang (GD-ZJ). DETAILED DESCRIPTION

[0045] The present application will be further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0046] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0047] The materials, reagents, etc. used in the following examples can be obtained commercially unless otherwise specified. Primer synthesis and sequencing were completed by Wuhan Tianyi Huiyuan Company Limited.

[0048] Example 1 Screening of SSR markers of Ptereleotris lunifasciata original species

[0049] The Ptereleotris lunifasciata genome was downloaded from Genbank with accession number PRJDB9369, total length 748.23 Mb, GC content 39.5%, scaffold N50 length 30.01 Mb, and contig N50 length 1.08 Mb. The SSR sequence was predicted using misa software, and SSR with 3-4 repeat units was selected. The SSR site with the highest expected heterozygosity was calculated.

[0050] The screened SSR site marker numbers are: PLWZ-01, PLWZ-02, PLWZ-03, PLWZ-04, PLWZ-05, and PLWZ-06, all of which are successfully amplified in Ptereleotris lunifasciata samples, have good polymorphism, and can clearly distinguish alleles.

[0051] Using primer3 software, the screened SSR was batch designed for PCR amplification product length of 200-300 bp primers. The primers were filtered, requiring a free energy greater than 30 (no hairpin structure). Using the filtered primers, genomic Blast comparison was performed, and specific primers were selected that theoretically have no sequence amplification in non-SSR regions and can be amplified in SSR regions. The SSR amplification fragment was selected for high polymorphism, good and stable amplification, high heterozygosity, different amplification fragment sizes, and the same or close annealing temperature (58-60℃), GC value control between 50%-60%.

[0052] The primers screened are shown in Table 1 below. After PCR amplification detection, the PCR reaction system is 25 μL, including: 10×PCR buffer without Mg 2+ 2.5 μL, 25 mM MgCl2 2.0 μL, 10 mM dNTP 0.5 μL, high-fidelity PCR enzyme 1 U, 10 μM forward primer 0.5 μL, 10 μM reverse primer 0.5 μL, DNA template 12.5 ng, and the rest is supplemented with sterile double distilled water to 25 μL. The reaction program of PCR amplification is preferably: 95 ℃ pre-denaturation for 5 minutes; 95 ℃ denaturation for 30 seconds, 60 ℃ annealing for 30 seconds, 72 ℃ extension for 30 seconds, a total of 35 cycles; 72 ℃ re-extension for 6 minutes.

[0053] Table 1 Primer set sequence list of SSR marker site

[0054]

[0055]

[0056] The PCR verification result shows that the primers in Table 1 can all successfully amplify in the Plectryagus leucopterus samples, and the bands are clear, stable in amplification, and good in repeatability.

[0057] Example 2 Verification of polymorphism of SSR marker site

[0058] A total of 173 Plectryagus leucopterus were collected from the South China Sea, the Philippines, Malaysia, Indonesia, and Australian waters, including Guangxi Weizhou (GX-WZ), Hainan Dongfang (HN-DF), Hainan Sanya (HN-SY), Hainan Wenchang (HN-WC), Guangdong Zhanjiang (GD-ZJ) in China, and Malaysia (GW-Ma), Indonesia (GW-In), the Philippines (GW-Ph), and Australia (GW-Au). Among them, 36 Plectryagus leucopterus were collected from the waters of Weizhou Island in Guangxi. The genomic DNA of Plectryagus leucopterus samples was extracted using the Tiangen Marine Animal Tissue Genomic DNA Extraction Kit; the extracted genomic DNA was used as a template, and 6 pairs of primers shown in Table 1 in Example 1 were used for PCR amplification of 173 samples, and the reaction system was 25 μL, including: 10×PCR buffer without Mg 2+ 2.5 μL, 25 mM MgCl2 2.0 μL, 10 mM dNTP 0.5 μL, high-fidelity PCR enzyme 1 U, 10 μM forward primer 0.5 μL, 10 μM reverse primer 0.5 μL, DNA template 12.5 ng, and the rest was supplemented with sterile double distilled water to 25 μL.

[0059] The PCR amplification reaction procedure is preferably as follows: pre-denaturation at 95℃ for 5 minutes; denaturation at 95℃ for 30 seconds, annealing at 60℃ for 30 seconds, extension at 72℃ for 30 seconds, for a total of 35 cycles; and re-extension at 72℃ for 6 minutes.

[0060] After the PCR amplification, capillary electrophoresis typing is performed, the 5' end of the primers of the 6 pairs of polymorphic SSR markers in Table 1 is labeled with a FAM fluorescent group, and capillary electrophoresis typing is performed using an ABI 3730XL genotype typing sequencer.

[0061] The capillary electrophoresis results of the 173 samples are subjected to peak chart interpretation and allele matrix construction using GeneMarker 4.0, genetic distance calculation is performed using GenALEx, and a cluster tree is constructed using MEGA 6.0 in the UPGMA method. The results are shown in Figure 1 As shown in the table, 39 samples including 36 samples collected from Weizhou Island in Guangxi are clustered in one clade, indicating that this branch is the original population of Weizhou Island.

[0062] The verification results of the polymorphism of the SSR marker sites are shown in Table 2, indicating that the six SSR sites used in the application have high polymorphism, but low polymorphism in the original population of Weizhou Island in Guangxi. Among them, the PLWZ-01 locus allele of the 36 samples of the original population of Ptereleotris punctatum in Weizhou Island in Guangxi is 258 / 261 / 267; the PLWZ-02 locus allele is 195 / 279 / 282 / 288; the PLWZ-03 locus allele is 238 / 251 / 256 / 279; the PLWZ-04 locus allele is 237 / 252 / 255 / 258; the PLWZ-05 locus allele is 257 / 272 / 291; and the PLWZ-06 locus allele is 178 / 199 / 215 / 219.

[0063] Table 2 Comparison of genetic diversity of the original population of Ptereleotris punctatum in Weizhou Island in Guangxi and other populations

[0064]

[0065] Note: N: sample number, Na: number of alleles, Ne: number of effective alleles, I: Shannon information index, Ho: observed heterozygosity, He: expected heterozygosity, F: fixation index, Pop: population, Mean: average value, SE: standard error.

[0066] Example 3 A method for identifying the original species of Ptereleotris punctatum in the sea area of Weizhou Island in Guangxi

[0067] Five Ptereleotris punctatum were collected from a Ptereleotris punctatum breeding farm in Xuwen, Zhanjiang, Guangdong, numbered, and sampled from the dorsal fin or caudal fin without causing obvious damage to the fish. The genomic DNA of Ptereleotris punctatum samples was extracted using the Tiangen Marine Animal Tissue Genomic DNA Extraction Kit. The extracted genomic DNA was used as a template to perform PCR amplification using the primer pairs PLWZ-01-F / R, PLWZ-02-F / R, PLWZ-03-F / R, PLWZ-04-F / R, PLWZ-05-F / R, and PLWZ-06-F / R shown in Table for different SSR marker sites PLWZ-01, PLWZ-02, PLWZ-03, PLWZ-04, PLWZ-05, and PLWZ-06 in Example 1.

[0068] The reaction system for PCR amplification was 25 μL, including: 10×PCR buffer 2.5 μL without Mg 2+ , 25 mM MgCl2 2.0 μL, 10 mM dNTP 0.5 μL, high-fidelity PCR enzyme 1 U, 10 μM forward primer 0.5 μL, 10 μM reverse primer 0.5 μL, DNA template 12.5 ng, and the rest was made up to 25 μL with sterile double distilled water.

[0069] The reaction program for PCR amplification was preferably: 95°C pre-denaturation for 5 minutes; 95°C denaturation for 30 seconds, 60°C annealing for 30 seconds, 72°C extension for 30 seconds, a total of 35 cycles; and 72°C for an additional 6 minutes of extension.

[0070] After PCR amplification, capillary electrophoresis typing was performed. The 5' ends of the 6 pairs of polymorphic SSR marker primers in Table 1 were labeled with FAM fluorescent groups, and capillary electrophoresis typing was performed using an ABI 3730XL genotype typing sequencer.

[0071] The capillary electrophoresis results were analyzed using GeneMarker 4.0 for peak interpretation and allele matrix construction, and the genetic distance was calculated using GenALEx. The clustering tree was constructed using MEGA 6.0 with the UPGMA method, and the obtained typing results were analyzed for peak interpretation.

[0072] The typing and identification results are shown in Table 3. Compared with the specific alleles of the Ptereleotris punctatum native population on Weizhou Island in Guangxi in Example 2, it was shown that No. 1 and No. 3 parent fish were native to Weizhou Island in Guangxi, and No. 2, No. 4, and No. 5 parent fish were not native to Weizhou Island in Guangxi. Therefore, No. 1 and No. 3 parent fish can be used as the breeding parents for Ptereleotris punctatum artificial propagation and release fry in Weizhou Island, Guangxi.

[0073] Table 3 Comparison of SSR site characteristics of Ptereleotris punctatum in Xuwen breeding farm and Ptereleotris punctatum native to Weizhou Island

[0074]

[0075] Example 4 Identification of Ptereleotris luniceps native species in Weizhou Island, Guangxi

[0076] A Ptereleotris luniceps was captured in Beihai sea area of Guangxi, and the population origin of the Ptereleotris luniceps was unknown. The Ptereleotris luniceps sample DNA was extracted, and the genomic DNA extracted was used as a template to perform PCR amplification on the primer pairs PLWZ-01-F / R, PLWZ-02-F / R, PLWZ-03-F / R, PLWZ-04-F / R, PLWZ-05-F / R and PLWZ-06-F / R for different SSR marker sites PLWZ-01, PLWZ-02, PLWZ-03, PLWZ-04, PLWZ-05 and PLWZ-06 shown in the table in Example 1.

[0077] The reaction system for PCR amplification was 25 μL, including: 10×PCR buffer 2.5 μL without Mg 2+ , 25 mM MgCl2 2.0 μL, 10 mM dNTP 0.5 μL, high-fidelity PCR enzyme 1 U, 10 μM forward primer 0.5 μL, 10 μM reverse primer 0.5 μL, DNA template 12.5 ng, and the rest was supplemented with sterile double distilled water to 25 μL.

[0078] The reaction program for PCR amplification was preferably: 95 °C pre-denaturation for 5 minutes; 95 °C denaturation for 30 seconds, 60 °C annealing for 30 seconds, 72 °C extension for 30 seconds, a total of 35 cycles; and 72 °C re-extension for 6 minutes.

[0079] After the PCR amplification was completed, capillary electrophoresis typing was performed, the 5' end of the 6 pairs of polymorphic SSR marker primers in Table 1 was labeled with a FAM fluorescent group, and capillary electrophoresis typing was performed using an ABI 3730XL genotype typing sequencer. The data of 5 samples from Weizhou Island in Guangxi, 5 samples from Zhanjiang in Guangdong, 10 samples from Wenchang in Hainan, 5 samples from Dongfang in Hainan, 5 samples from Sanya in Hainan, and 5 samples from Wenchang in Hainan were combined and analyzed, the genetic distance was calculated using GenALEx, the clustering tree was constructed using MEGA6.0 with the UPGMA method, and the peak chart of the obtained typing results was interpreted.

[0080] The results are shown in Figure 2 , the Ptereleotris luniceps sample was clustered with the samples from Weizhou Island in Guangxi, and the sample was identified as the native species of Weizhou Island in Guangxi.

[0081] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are included in the protection scope of the present application. SEQUENCE LISTING <110> Guangdong Ocean University Institute of Southern Oceanography, Chinese Academy of Sciences <120> A set of SSR molecular marker primers for identifying the original species of Ptereleotris rubripectoralis in Weizhou Island, Guangxi, and application thereof <160> 18 <170> SIPOSequenceListing 1.0 <210> 1 <211> 300 <212> DNA <213> PLWZ-01 (SIPOSequenceListing 1.0) <400> 1 gctgaccggt gagtctcaac ttttgttctc aacttttatt tatttattta taataataat 60 aataataata ataataataa taataataat aataataata ataataataa taataataat 120 aataattgtg aataattaat ttacataaca ataataataa atattattat tattaattaa 180 tgtattaatg ttgtttggtg actacactaa agtttaaagt ctcagttcgg gttataaagg 240 ttgttttttt gtttttctca cagcggtcag gccgtggccg ccatcatctt cgtgctcggt 300 <210> 2 <211> 233 <212> DNA <213> PLWZ-02 (SIPOSequenceListing 1.0) <400> 2 gtgctttgct cagcttgctt accccacaac ccgactccgg caaagtggat gaaatggatg 60 gctggatgga tggatggata aaaataataa taataataat aataataata ataataataa 120 taataataat aataataata ataataataa taataataag catacacagt tagaaagtac 180 acactcacac tcacacaccc aagtaacgat aagtgtgtcc ctgagcaaag gtc 233 <210> 3 <211> 258 <212> DNA <213> PLWZ-03 (SIPO SequenceListing 1.0) <400> 3 gctgtgtttg agcaaagcca aaataaaatt tccactgagc tggacaataa agtctatcta 60 tctatctatc tatctatcta tctatctaat aacaataata ataataataa taataataat 120 aataataata ataataataa taataataat aataataata ataataatag taataataat 180 gatgataata ataataataa taataataat aataataata aattctattt attagcgcct 240 ttccggacac cagaggtc 258 <210> 4 <211> 288 <212> DNA <213> PLWZ-04 (SIPO SequenceListing 1.0) <400> 4 ggtgaccttg agtgtcctga aaggtgccaa taaataacat tttataataa taataataat 60 aataataata ataataataa taataataat aataataatt attattatta ttattattgt 120 tgttgttgtt gttgttattg ttgttattgt taatattatt attattatta ttattattat 180 tattattatt attattatta ttattattat tattattata aatagggata caatgaaggt 240 atattggttt gcatgataat aattgacagt gtgtacaaca gctggagc 288 <210> 5 <211> 296 <212> DNA <213> PLWZ-05 (SIPO SequenceListing 1.0) <400> 5 tggctttggc tcaaatgaca tttttctaca gctaatgaac tgatgcacct gcaatgctaa 60 taataataat aataataata ataataataa taataataat aataataata ataataataa 120 taataataat aataataata ataacaacaa taaagttgtt tctttttttt taaaaaagtc 180 cagttgctat ttatatttcg ttaaccacaa tgttgtgggg ttttttcttt taccacaaat 240 agaaagaaga ttcagaattt tacttccctc atttattccc tgtcagtttc cttcca 296 <210> 6 <211> 217 <212> DNA <213> PLWZ-06 (SIPO SequenceListing 1.0) <400> 6 gccctacatt ctcatcgcag agttaataaa ttgcatatta ttattattat tattattatt 60 attattatta ttattattat tattattatt attattatta tctctgacat gaagactggg 120 cagctgaaac ccgatctctg tcaacccaaa catttccaag gcatttgttt ttctggatag 180 tatttgattt ccccacattc cctgaacgta gcagcag 217 <210> 7 <211> 20 <212> DNA <213> PLWZ-01-F (SIPO SequenceListing 1.0) <400> 7 gctgaccggt gagtctcaac 20 <210> 8 <211> 20 <212> DNA <213> PLWZ-01-R (SIPO SequenceListing 1.0) <400> 8 accgagcacg aagatgatgg 20 <210> 9 <211> 20 <212> DNA <213> PLWZ-02-F (SIPO SequenceListing 1.0) <400> 9 gtgctttgct cagcttgctt 20 <210> 10 <211> 20 <212> DNA <213> PLWZ-02-R (SIPO SequenceListing 1.0) <400> 10 gacctttgct cagggacaca 20 <210> 11 <211> 20 <212> DNA <213> PLWZ-03-F (SIPO SequenceListing 1.0) <400> 11 gctgtgtttg agcaaagcca 20 <210> 12 <211> 20 <212> DNA <213> PLWZ-03-R (SIPO SequenceListing 1.0) <400> 12 gacctctggt gtccggaaag 20 <210> 13 <211> 20 <212> DNA <213> PLWZ-04-F (SIPO SequenceListing 1.0) <400> 13 gcattgagca tcctggcaag 20 <210> 14 <211> 20 <212> DNA <213> PLWZ-04-R (SIPO SequenceListing 1.0) <400> 14 cagatcagga cagcactccg 20 <210> 15 <211> 20 <212> DNA <213> PLWZ-05-F (SIPO SequenceListing 1.0) <400> 15 tggctttggc tcaaatgaca 20 <210> 16 <211> 20 <212> DNA <213> PLWZ-05-R (SIPO SequenceListing 1.0) <400> 16 tggaaggaaa ctgacaggga 20 <210> 17 <211> 21 <212> DNA <213> PLWZ-06-F (SIPO SequenceListing 1.0) <400> 17 gccctacatt ctcatcgcag a 21 <210> 18 <211> 20 <212> DNA <213> PLWZ-06-R (SIPO SequenceListing 1.0) <400> 18 ctgctgctac gttcagggaa 20

Claims

1. An SSR molecular marker for identifying the native species of the leopard-gill spiny perch of Weizhou Island, Guangxi, characterized in that, The SSR molecular markers are PLWZ-01, PLWZ-02, PLWZ-03, PLWZ-04, PLWZ-05, and PLWZ-06, and their nucleotide sequences are shown in SEQ ID NO.1 to 6, respectively.

2. A primer set for amplifying the SSR molecular marker of claim 1, characterized in that, The primer set is as follows: The primer sequences for amplifying PLWZ-01 are shown in SEQ ID NO.7-8, respectively; The primer sequences for amplifying PLWZ-02 are shown in SEQ ID NO.9-10, respectively; The primer sequences for amplifying PLWZ-03 are shown in SEQ ID NO.11-12, respectively; The primer sequences for amplifying PLWZ-04 are shown in SEQ ID NO.13-14, respectively; The primer sequences for amplifying PLWZ-05 are shown in SEQ ID NO.15-16, respectively; The primer sequences for amplifying PLWZ-06 are shown in SEQ ID NO.17-18, respectively.

3. The application of the reagent for detecting the SSR molecular marker described in claim 1, or the primer set described in claim 2, in identifying the native species of *Leopard gill sea bass* from Weizhou Island, Guangxi, or in preparing a detection kit for identifying the native species of *Leopard gill sea bass* from Weizhou Island, Guangxi, characterized in that... The alleles for identifying the native species of the leopard-spotted spiny perch on Weizhou Island, Guangxi, are as follows: The PLWZ-01 sites are: 258, 261, and 267; The PLWZ-02 sites are: 195, 279, 282, and 288. The PLWZ-03 sites are: 238, 251, 256, and 279; The PLWZ-04 sites are: 237, 252, 255, and 258. The PLWZ-05 sites are: 257, 272, and 291; The PLWZ-06 sites are: 178, 199, 215, and 219.

4. A detection kit for identifying the native species of the leopard-gill spiny perch of Weizhou Island, Guangxi, characterized in that, Contains the SSR molecular marker of claim 1 or the primer set of claim 2.

5. A method for identifying the native species of the leopard-gill spiny perch of Weizhou Island, Guangxi, characterized in that, Includes the following steps: S1. Collect samples of the leopard gill-tailed spiny perch population to be identified and extract DNA from the samples; S2. Using the DNA extracted in step S1 as a template, perform PCR amplification using the primer set described in claim 2; S3. Perform capillary electrophoresis to characterize the PCR products amplified in step S2; S4. Perform peak plot interpretation and construct allele matrix on the typing results obtained in step S3; S5. The alleles of the leopard-gill sea bass to be tested obtained through step S4 are compared with the alleles of the known native species of leopard-gill sea bass on Weizhou Island, Guangxi, and the genetic distance between the samples is calculated. The alleles of the native species of the leopard-gill spiny perch from Weizhou Island, Guangxi are: The PLWZ-01 sites are: 258, 261, and 267; The PLWZ-02 sites are: 195, 279, 282, and 288. The PLWZ-03 sites are: 238, 251, 256, and 279; The PLWZ-04 sites are: 237, 252, 255, and 258. The PLWZ-05 sites are: 257, 272, and 291; The PLWZ-06 sites are: 178, 199, 215, and 219; S6. Perform cluster analysis on the genetic distance obtained in step S5, construct a cluster tree, and use the cluster tree to determine whether the leopard gill spiny perch to be detected is a native species of Weizhou Island.

6. The method according to claim 5, characterized in that, The reaction system for PCR amplification in step S2 is: free of Mg. 2+ The following ingredients were added: 2.5 μL of 10×PCR buffer, 2.0 μL of 25 mM MgCl2, 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, and 12.5 ng of DNA template. The remainder was made up to 25 μL with sterile double-distilled water.

7. The method according to claim 5, characterized in that, The PCR amplification reaction program in step S2 is as follows: 95℃ pre-denaturation for 5 minutes; 95℃ denaturation for 30 seconds, 60℃ annealing for 30 seconds, 72℃ extension for 30 seconds, for a total of 35 cycles; 72℃ extension for 6 minutes.

8. The method according to claim 5, characterized in that, In step S3, capillary electrophoresis typing involves labeling the 5' end of the primer set used in step S2 with the FAM fluorescent group and performing capillary electrophoresis typing using an ABI 3730XL genotyping sequencer.

9. The application of the method according to any one of claims 5 to 8 in identifying the original parent species of leopard gill spiny perch in geographical populations or in the identification of seedlings for propagation and release, characterized in that, The leopard-gill spiny perch geographical population or the seedlings released for propagation are native seedlings of the leopard-gill spiny perch from Weizhou Island, Guangxi.

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