Epinephelus coioides aquaculture ssr marker, detection primer and application thereof

Abstract

The application discloses a Epinephelus coioides aquaculture SSR marker, a detection primer thereof and application. The SSR marker comprises ECDSSR-01, ECDSSR-02, ECDSSR-03, ECDSSR-04, ECDSSR-05, ECDSSR-06, ECDSSR-07 and ECDSSR-08. The application is based on the genome data to develop the SSR marker and the primer, the developed SSR marker has higher polymorphism than the marker developed in the prior art, genetic diversity analysis is carried out on 9 individuals, and more than 12 alleles are obtained, which is much higher than the level of 4 to 12 alleles of 20 individuals in the prior art, and has an advantage when evaluating a population with low genetic diversity (such as a proliferation and release population). The developed SSR molecular marker can be applied to the field of genetic diversity analysis of the Epinephelus coioides and the like, and provides a theoretical basis for future investigation of Epinephelus coioides germplasm resources and evaluation of release effect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biology, and particularly relates to a SSR marker, primer and method for efficiently evaluating the genetic diversity of Epinephelus coioides populations based on the genome of Epinephelus coioides, and application of the SSR marker, primer and method in the aquaculture of Epinephelus coioides. BACKGROUND

[0002] Epinephelus coioides, commonly known as "green spot", belongs to Perciformes, Serranidae and Epinephelus. Epinephelus coioides is an important farmed fish in the south of China. Due to its delicious meat, rich nutrition, strong resistance, fast growth, and bright color, the market price is high and stable, and it is favored by consumers and breeders.

[0003] However, due to overfishing, water pollution and other human factors in recent years, the wild population resources and genetic diversity of Epinephelus coioides have decreased, and it is necessary to protect and reasonably develop the germplasm resources through artificial aquaculture. At the same time, the genetic diversity of Epinephelus coioides needs to be evaluated in the process of natural resource assessment, artificial breeding and restocking.

[0004] Among genetic molecular markers, three kinds of markers, sequence molecular markers, simple sequence repeats (SSR) and single nucleotide polymorphism (SNP) are frequently used at present, each of which has its own advantages and disadvantages. SSR (Simple Sequence Repeats) marker is a molecular marker technology based on specific primer PCR developed in recent years, also known as microsatellite DNA, which is a kind of tandem repeat sequence composed of several nucleotides (usually 1-6) as repeating units, with a length of several dozen nucleotides. Microsatellites are randomly distributed on chromosomes and evenly distributed in eukaryotic genomes in multiple copies. The polymorphism is caused by different number of repeats and incomplete degree of repeats. For a known microsatellite DNA sequence, the repeat sequence region has a relatively high mutation rate (10 2 -10 6 times per generation), thereby generating high levels of allelic polymorphism, and the flanking sequence at both ends of the repeat region is relatively conservative. Therefore, specific primers can be designed using the flanking sequence to amplify the site for related polymorphism research.

[0005] The development methods of SSR molecular markers include: genomic enzyme cutting hybridization method, random amplification hybridization method, anchor PCR amplification method, enrichment library separation method, AFLP-based SSR rapid separation method and EST-SSR method based on transcriptome sequencing. At present, the most widely used is the high-throughput and simple-to-operate EST-SSR method. However, the SSR tags obtained by the EST-SSR method are located in the transcription region, which is a region with low SSR density on the genome, and may be positively selected with trait association, and cannot completely and accurately reflect the neutral population gene flow.

[0006] In the past, the number of alleles of the SSR marker of Epinephelus coioides was 4-12, and the use efficiency was limited when evaluating the population with low genetic diversity (such as artificial breeding population, release population). With the development of whole genome sequencing technology, the SSR marker with the highest expected number of alleles can be obtained by directly screening and analyzing the SSR sequence in the genome. Therefore, we hope to mine the efficient SSR marker in the genome of Epinephelus coioides and develop primers and detection methods, so as to lay a foundation for efficient evaluation of genetic diversity of Epinephelus coioides and provide breeding benefits. SUMMARY

[0007] The first object of the present application is to provide a SSR marker for efficiently evaluating the genetic diversity of the population of Epinephelus coioides, which has strong polymorphism and high heterozygosity.

[0008] The SSR marker of the present application has the marker number of ECDSSR-01, ECDSSR-02, ECDSSR-03, ECDSSR-04, ECDSSR-05, ECDSSR-06, ECDSSR-07 and ECDSSR-08, and the specificities are as follows:

[0009] The nucleotide sequence of ECDSSR-01 is shown as SEQ ID NO. 1;

[0010] The nucleotide sequence of ECDSSR-02 is shown as SEQ ID NO. 2;

[0011] The nucleotide sequence of ECDSSR-03 is shown as SEQ ID NO. 3;

[0012] The nucleotide sequence of ECDSSR-04 is shown as SEQ ID NO. 4;

[0013] The nucleotide sequence of ECDSSR-05 is shown as SEQ ID NO. 5;

[0014] The nucleotide sequence of ECDSSR-06 is shown as SEQ ID NO. 6;

[0015] The nucleotide sequence of the ECDSSR-07 is shown as SEQ ID NO. 7.

[0016] The nucleotide sequence of the ECDSSR-08 is shown as SEQ ID NO. 8.

[0017] The second object of the present application is to provide a SSR marker detection primer for efficiently evaluating the genetic diversity of Epinephelus coioides population, which has stable amplification and good repeatability.

[0018] For ECDSSR-01 site:

[0019] ECDSSR-01-F: 5'-GTCCTCTGCTTTGGGCTGAA-3';

[0020] ECDSSR-01-R: 5'-GAGTCACTGCACACTGACGA-3';

[0021] For ECDSSR-02 site:

[0022] ECDSSR-02-F: 5'-CCTGTTGGCTGACTTTGAGC-3';

[0023] ECDSSR-02-R: 5'-CTGGAAGGCCTGTTCAGTCA-3';

[0024] For ECDSSR-03 site:

[0025] ECDSSR-03-F: 5'-TACTGCAGCACCACAGACTG-3';

[0026] ECDSSR-03-R: 5'-TGAACAGGCTGGTCTGCTTT-3';

[0027] For ECDSSR-04 site:

[0028] ECDSSR-04-F: 5'-ACGCCATGCATGTCCATGTA-3';

[0029] ECDSSR-04-R: 5'-AGAGCCGCTTGTTCAAGAGG-3';

[0030] For ECDSSR-05 site:

[0031] ECDSSR-05-F: 5'-AGGGTCTGCCTCTCCATCAT-3';

[0032] ECDSSR-05-R: 5'- AGACAGGGATCACTGCAAGC -3';

[0033] For ECDSSR-06 site:

[0034] ECDSSR-06-F: 5'- GGGACAGGTGAGCAGATCAG -3';

[0035] ECDSSR-06-R: 5'- CCCATGAGGTGTCACTAGTGG -3';

[0036] For ECDSSR-07 site:

[0037] ECDSSR-07-F: 5'- ATCCTTTATGCCAGGGCTGC -3';

[0038] ECDSSR-07-R: 5'- GCTCTTTGTCTGCCAACAGC -3';

[0039] For ECDSSR-08 site:

[0040] ECDSSR-08-F: 5'- GCTGTGGCTCCTCAGTCATT -3';

[0041] ECDSSR-08-R: 5'- ATTTCCCACTCAGCGTGTGT -3'.

[0042] Preferably, the 5' end of the forward primer of the detection primer is labeled with a fluorescent group, such as a FAM fluorescent group.

[0043] A third object of the present application is to provide the use of the reagent for detecting the above-mentioned SSR marker in the preparation of a product for analyzing the genetic diversity of a group of Epinephelus coioides and identifying a population.

[0044] A fourth object of the present application is to provide the use of the above-mentioned SSR marker or the above-mentioned detection primer in the preparation of a kit for analyzing the genetic diversity of a group of Epinephelus coioides and identifying a population.

[0045] A fifth object of the present application is to provide a kit for analyzing the genetic diversity of a group of Epinephelus coioides and identifying a population, which comprises the above-mentioned detection primer.

[0046] A sixth object of the present application is to provide a method for efficiently evaluating the genetic diversity of a group of Epinephelus coioides, comprising the following steps:

[0047] (1) Collecting Epinephelus coioides population sample, extracting individual DNA of Epinephelus coioides;

[0048] (2) Using the detection primer to carry out PCR amplification with the genomic DNA extracted in step (1) as a template;

[0049] (3) Carrying out typing on the PCR product amplified in step (2);

[0050] (4) Carrying out genetic diversity analysis on the typing result obtained in step (3).

[0051] Preferably, the PCR amplification in step (2) is carried out in a reaction system of 25 μL, including: 10×PCRbuffer 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 is supplemented with sterile double distilled water to 25 μL.

[0052] The reaction procedure is as follows: 95ºC pre-denaturation for 5 minutes; 95ºC denaturation for 30 seconds, 60ºC annealing for 30 seconds, 72ºC extension for 30 seconds, totally 35 cycles; and 72ºC re-extension for 6 minutes.

[0053] Preferably, the typing in step (3) is carried out by marking the 5' end of the forward primer of the 8 pairs of polymorphic SSR marker primers with FAM fluorescent groups, and carrying out capillary electrophoresis typing by using an ABI 3730XL genotyping sequencer.

[0054] Preferably, the genetic diversity analysis in step (4) is carried out by using GenAIex software to carry out genetic diversity analysis, including the commonly used indexes reflecting population genetic diversity such as the number of alleles (Na) of each locus, the effective number of alleles (Ne), the Shannon information index (I), the observed heterozygosity (Ho), the expected heterozygosity (He), and the fixation index (F) is used to measure the degree of deviation of the observed heterozygosity from the Hardy-Weinberg equilibrium, and MEGA6.0 is used to construct a clustering tree by using the UPGMA method.

[0055] The last purpose of the present application is to provide the application of the above-mentioned SSR marker, detection primer, kit or evaluation method in the genetic diversity analysis of Epinephelus coioides breeding population, the identification of geographical population or the evaluation of the effect of propagation and release, etc.

[0056] Compared with the prior art, the present application has the following advantages:

[0057] The application is based on the SSR marker and primer developed based on genomic data, and the developed SSR marker has higher polymorphism than the marker developed in the prior art, and more than 12 alleles are obtained by genetic diversity analysis of 9 individuals, which is much higher than the level of 4-12 alleles of 20 individuals in the prior art, and has an advantage in evaluating a population with low genetic diversity (such as a proliferation and release population). The SSR molecular marker developed in the application can be used in the field of genetic diversity analysis of Epinephelus coioides, and provides a theoretical basis for future investigation of Epinephelus coioides germplasm resources and evaluation of release effect. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 The genetic relationship diagram of 30 Epinephelus coioides in Zhuhai Doumen, Wanshan Archipelago and Yangjiang Hailing Island is analyzed by SSR, ZH: Zhuhai Doumen, WS: Wanshan Archipelago, YJ: Yangjiang Hailing Island DETAILED DESCRIPTION

[0059] The application will be further described below in combination with examples, but is not limited thereto.

[0060] In the following examples, the experimental methods are conventional methods or are carried out according to the instructions of the kit unless otherwise specified. The materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified. Primer synthesis and sequencing are completed by Wuhan Tianyi Huiyuan Company Limited.

[0061] The genome of Epinephelus coioides is downloaded from Genbank, the submission number is PRJEB28248, the total length is 1023.56 Mb, the GC content is 41.3%, the scaffold N50 length is 2.44 Mb, and the contig N50 length is 2.33 Mb. The SSR sequence is predicted using the misa software, the SSR with 3-4 repeat units is selected, and the SSR site with the highest expected heterozygosity is calculated according to the second-generation sequencing data.

[0062] Eight SSR sites with high polymorphism in the genome of Epinephelus coioides are obtained, and the marker numbers are ECDSSR-01, ECDSSR-02, ECDSSR-03, ECDSSR-04, ECDSSR-05, ECDSSR-06, ECDSSR-07 and ECDSSR-08.

[0063] The nucleotide sequence of ECDSSR-01 is shown as SEQ ID NO. 1.

[0064] The nucleotide sequence of ECDSSR-02 is shown as SEQ ID NO. 2.

[0065] The nucleotide sequence of ECDSSR-03 is shown as SEQ ID NO. 3.

[0066] The nucleotide sequence of the ECDSSR-04 is shown as SEQ ID NO. 4;

[0067] The nucleotide sequence of the ECDSSR-05 is shown as SEQ ID NO. 5;

[0068] The nucleotide sequence of the ECDSSR-06 is shown as SEQ ID NO. 6;

[0069] The nucleotide sequence of the ECDSSR-07 is shown as SEQ ID NO. 7;

[0070] The nucleotide sequence of the ECDSSR-08 is shown as SEQ ID NO. 8.

[0071] The primer3 software is used to design the PCR amplification product length of 200-300 bp of the screened SSR. The primer is filtered, and the free energy is required to be greater than 30 (no hairpin structure). The filtered primer is used for genome Blast comparison, and specific primers are selected, which theoretically have no sequence amplification in the non-SSR region, and can be amplified in the SSR region. The SSR amplification fragment is selected to have high polymorphism, good and stable amplification, high heterozygosity, different amplification fragment size, and the same or close annealing temperature (58-60ºC), and the GC value is controlled between 50%-60%. Through PCR amplification, the size of the amplification product is detected by capillary electrophoresis, and 8 pairs of specific amplification and high polymorphism SSR marker primers are screened, and the detection primer includes:

[0072] For ECDSSR-01 site:

[0073] ECDSSR-01-F: 5'-GTCCTCTGCTTTGGGCTGAA-3';

[0074] ECDSSR-01-R: 5'-GAGTCACTGCACACTGACGA-3';

[0075] For ECDSSR-02 site:

[0076] ECDSSR-02-F: 5'-CCTGTTGGCTGACTTTGAGC-3';

[0077] ECDSSR-02-R: 5'-CTGGAAGGCCTGTTCAGTCA-3';

[0078] For ECDSSR-03 site:

[0079] ECDSSR-03-F: 5'- TACTGCAGCACCACAGACTG -3';

[0080] ECDSSR-03-R: 5'- TGAACAGGCTGGTCTGCTTT -3';

[0081] For ECDSSR-04 site:

[0082] ECDSSR-04-F: 5'- ACGCCATGCATGTCCATGTA -3';

[0083] ECDSSR-04-R: 5'- AGAGCCGCTTGTTCAAGAGG -3';

[0084] For ECDSSR-05 site:

[0085] ECDSSR-05-F: 5'- AGGGTCTGCCTCTCCATCAT -3';

[0086] ECDSSR-05-R: 5'- AGACAGGGATCACTGCAAGC -3';

[0087] For ECDSSR-06 site:

[0088] ECDSSR-06-F: 5'- GGGACAGGTGAGCAGATCAG -3';

[0089] ECDSSR-06-R: 5'- CCCATGAGGTGTCACTAGTGG -3';

[0090] For ECDSSR-07 site:

[0091] ECDSSR-07-F: 5'- ATCCTTTATGCCAGGGCTGC -3';

[0092] ECDSSR-07-R: 5'- GCTCTTTGTCTGCCAACAGC -3';

[0093] For ECDSSR-08 site:

[0094] ECDSSR-08-F: 5'- GCTGTGGCTCCTCAGTCATT -3';

[0095] ECDSSR-08-R: 5'- ATTTCCCACTCAGCGTGTGT -3'.

[0096] The 5' end of the forward primer of the detection primer is labeled with a fluorescent group, such as a FAM fluorescent group.

[0097] A method for efficiently evaluating the genetic diversity of Epinephelus coioides populations, comprising the following steps

[0098] (1) Collecting samples of Epinephelus coioides populations, the sampling site being the dorsal fin or the tail fin, the sampling process not causing obvious damage to the fish, and extracting DNA of individual Epinephelus coioides;

[0099] (2) Using the genomic DNA extracted in step (1) as a template, and performing PCR amplification using the primer pair ECDSSR-01-F / R for the ECDSSR-01 site, the primer pair ECDSSR-02-F / R for the ECDSSR-02 site, the primer pair ECDSSR-03-F / R for the ECDSSR-03 site, the primer pair ECDSSR-04-F / R for the ECDSSR-04 site, the primer pair ECDSSR-05-F / R for the ECDSSR-05 site, the primer pair ECDSSR-06-F / R for the ECDSSR-06 site, the primer pair ECDSSR-07-F / R for the ECDSSR-07 site, and the primer pair ECDSSR-08-F / R for the ECDSSR-08 site, respectively;

[0100] The PCR amplification has a reaction system of 25 μL, comprising: 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 being supplemented with sterile double-distilled water to 25 μL.

[0101] The reaction program of the PCR amplification is preferably: 95ºC pre-denaturation for 5 minutes; 95ºC denaturation for 30 seconds, 60ºC annealing for 30 seconds, 72ºC extension for 30 seconds, a total of 35 cycles; and 72ºC re-extension for 6 minutes.

[0102] (3) Performing capillary electrophoresis typing on the PCR products amplified in step (2), which is capillary electrophoresis typing performed by an ABI 3730XL genotyping sequencer.

[0103] (4) Genetic diversity analysis is performed on the typing results obtained in step (3), specifically, genetic diversity analysis is performed by using GenAIex software, including the commonly used indexes reflecting population genetic diversity such as the number of alleles (Na) of each locus, the number of effective alleles (Ne), the Shannon information index (I), the observed heterozygosity (Ho), the expected heterozygosity (He), and the fixation index (F) for measuring the degree of deviation of the observed heterozygosity from the Hardy-Weinberg equilibrium, and MEGA6.0 is used to construct a clustering tree by the UPGMA method.

[0104] Example 1:

[0105] A total of 9 Epinephelus radiatus samples were collected from Doumen, Zhuhai, Wanshan Archipelago, and Yangjiang Hailing Island in Guangdong Province. The 8 pairs of detection primers for the above-mentioned ECDSSR-01, ECDSSR-02, ECDSSR-03, ECDSSR-04, ECDSSR-05, ECDSSR-06, ECDSSR-07, and ECDSSR-08 loci were used to perform PCR amplification analysis and capillary electrophoresis typing on the 9 individuals, and statistical analysis was performed by software, and the results are shown in Table 1.

[0106] The number of alleles (Na) of each SSR marker is between 12 and 13, with an average of 12.375 alleles per SSR marker, the number of effective alleles is between 6.75 and 10.8, with an average of 8.944, the Shannon information index is between 2.289 and 2.476, with an average of 2.364, the observed heterozygosity is between 0.667 and 1.000, with an average of 0.861, the expected heterozygosity is between 0.852 and 0.907, with an average of 0.886, the observed heterozygosity is close to the expected heterozygosity, indicating that there is no heterozygosity loss, and the fixation index is between -0.141 and 0.26, with an average of 0.027.

[0107] Table 1. Genetic diversity parameters of 8 SSRs in 9 individuals of Epinephelus radiatus from different sources

[0108] Site name N Na Ne I Ho He F ECDSSR-01 9 12.000 9.529 2.370 1.000 0.895 -0.117 ECDSSR-02 9 12.000 9.529 2.370 0.889 0.895 0.007 ECDSSR-03 9 13.000 6.750 2.293 0.778 0.852 0.087 ECDSSR-04 9 12.000 10.125 2.399 0.667 0.901 0.260 ECDSSR-05 9 12.000 9.000 2.351 0.889 0.889 0.000 ECDSSR-06 9 13.000 10.800 2.476 0.778 0.907 0.143 ECDSSR-07 9 13.000 8.100 2.366 1.000 0.877 -0.141 ECDSSR-08 9 12.000 7.714 2.289 0.889 0.870 -0.021

[0109] 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

[0110] Example 2:

[0111] Collect 3 groups of Epinephelus coioides from Dongfang, Hainan, 10 individuals in each group, use 8 pairs of detection primers for ECDSSR-01, ECDSSR-02, ECDSSR-03, ECDSSR-04, ECDSSR-05, ECDSSR-06, ECDSSR-07, ECDSSR-08 sites respectively to conduct PCR amplification analysis and capillary electrophoresis typing on 30 individuals in total of 3 groups, and use software for statistical analysis, and the results are shown in Table 2.

[0112] The average allele numbers of 3 groups of Epinephelus coioides from Dongfang, Hainan are 5.25, 4.25, 5.25 respectively, the average effective allele numbers are 3.711, 3.537, 4.204 respectively, the average Shannon information indexes are 1.425, 1.311, 1.506 respectively, the average observed heterozygosity are 0.688, 0.763, 0.763 respectively, the average expected heterozygosity are 0.725, 0.704, 0.757 respectively, and the evaluation fixation indexes are 0.051, -0.079, -0.003 respectively.

[0113] Table 2. Genetic diversity parameters of 3 groups of Epinephelus coioides from Dongfang, Hainan

[0114] Pop N Na Ne I Ho He F Population 1 Mean 10 5.250 3.711 1.425 0.688 0.725 0.051 SE 0.313 0.205 0.050 0.035 0.014 0.045 Population 2 Mean 10 4.250 3.537 1.311 0.763 0.704 -0.079 SE 0.250 0.275 0.072 0.046 0.025 0.043 Population 3 Mean 10 5.250 4.204 1.506 0.763 0.757 -0.003 SE 0.250 0.251 0.051 0.060 0.013 0.068

[0115] N: sample number, Na: allele number, Ne: effective allele number, I: Shannon information index, Ho: observed heterozygosity, He: expected heterozygosity, F: fixation index, Pop: population, Mean: average value, SE: standard error

[0116] Example 3:

[0117] Collect 10 individuals of Epinephelus coioides from Doumen, Zhuhai, Wanshan Islands, Yangjiang Hailing Island, Guangdong Province, in total 30 individuals, use 8 pairs of detection primers for ECDSSR-01, ECDSSR-02, ECDSSR-03, ECDSSR-04, ECDSSR-05, ECDSSR-06, ECDSSR-07, ECDSSR-08 sites respectively to conduct PCR amplification analysis and capillary electrophoresis typing, use GeneMarker4.0 to interpret peak chart and establish allele matrix, use GenALEx to calculate genetic distance, and use MEGA6.0 to construct clustering tree by UPGMA method.

[0118] As Figure 1The genetic relationship of Epinephelus coioides collected from different sea areas was consistent with the geographical distribution by SSR analysis. The genetic distance between the population of E. coioides collected from Doumen of Zhuhai and the population of E. coioides collected from Wanshan Islands was relatively close, and the two populations were mixed. The genetic distance between the population of E. coioides collected from Yangjiang and the other two populations was relatively far, and the two populations were significantly separated. SEQUENCE LISTING <110> INSTITUTE OF OCEANOGRAPHY, CHINESE ACADEMY OF SCIENCES <120> A SSR marker for Epinephelus coioides aquaculture and detection primer and application thereof <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 297 <212> DNA <213> Epinephelus coioides <400> 1 gagtcactgc acactgacga gttcaaaaca ctcgagtcaa cttcctgtgt gtaattacag 60 tgtcgtgatt aaaatgtgag tctgtgttca tccatgttgt tggagtttta cctgctccag 120 cttcatcaga gaagggtcag agggtcagag gtcaggggtc agaggtctgg agctggaggg 180 agttcttctt cttcttcttc ttcttcttct tcttcttctt cttcttcttc ttcttcttca 240 gctccaacca caggcatgtt aattagcagt gaggcatttc agcccaaagc agaggac 297 <210> 2 <211> 300 <212> DNA <213> Epinephelus coioides <400> 2 ctggaaggcc tgttcagtca tccatccatg acaaaacaga tcacaggtat aacattacaa 60 cagtaagaga gaaagagaca gaaaacaaac attaagtcaa cccatggtaa acaaaacaa 120 aaaaaaagga aaaagacatt caaattgtat tttctctatc tatctatcta tctatctatc 180 tatctatcta tctatctatc tatctatcta tctatctatc tatctatcta tctatctgag 240 ttcagactgc tattgttctg tgactctcct gtcatctaca gctcaaagtc agccaacagg 300 <210> 3 <211> 297 <212> DNA <213> Zebrafish (Danio rerio) <400> 3 tgaacaggct ggtctgcttt atgagtactt ttactttaaa tacttaatta tattgtggtg 60 acattacttt aatttcagta aaatcttctt cttcttcttc tcttcttctt ctcttcttct 120 tcttcttctc ttcttcttct tcttcttctt cttcttcttc ttcttcttct tcttcttctt 180 cttcttcttt tcttcttctt cttcttcttc ttcttctctt ctttcttctt ctcttttctt 240 cttcttcttt cttcttgctt cttcctgctc ttcttaccag tctgtggtgc tgcagta 297 <210> 4 <211> 269 <212> DNA <213> Zebrafish (Danio rerio) <400> 4 agagccgctt gttcaagagg gtagtgattg tagaaagaag aaagtattat gatgctgctc 60 aatacaacca gtttaaattt aataataata ataataataa taataataat aataataata 120 ataataataa taataataat attatatgga attgccctat ggcttcactg agacctctta 180 ccattcctgt acaatccacc taaaacacct attcagggca tacctgaagt aaattgacag 240 ctgttcttgt acatggacat gcatggcgt 269 <210> 5 <211> 212 <212> DNA <213> Zebrafish (Danio rerio) <400> 5 agacagggat cactgcaagc atgcacgaac attagaaaat gggagataga tagatagata 60 gatagataga tagatagata gatagataga tagatagata gatagataga tagatcaaac 120 ccgccttact gtctcagcag ggatgtgccc ccctttgtgc ctccgcggag gccttggccg 180 aacccgtgtc acatgatgga gaggcagacc ct 212 <210> 6 <211> 275 <212> DNA <213> Zebrafish (Danio rerio) <400> 6 GAGAAGAAGA GAAGAAGAAG AAGAAGAAGA AGAAGAAGAAGAAGAAG 60 AAGAAGAAGA AGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAG 120 AAGAAGAAGA AGAAGAAGAAGAAGAAGAAGAAGAAGAAG 180 AAGAAGAAGA AGAAGAAGAAGAAGAAGAAGAAGAAG 240 AAGAAGAAGA AGAAGAAGAAGAAGAAGAAGAAG 275 <210> 7 <211> 262 <212> DNA <213> Zebrafish (Danio rerio) <400> 7 GAGAAGAAGA GAAGAAGAAG AAGAAGAAGA AGAAGAAGAAGAAGAAG 60 AAGAAGAAGA AGAAGAAGAAGAAGAAGAAGAAGAAGAAG 120 AAGAAGAAGA AGAAGAAGAAGAAGAAGAAGAAGAAG 180 AAGAAGAAGA AGAAGAAGAAGAAGAAGAAGAAG 240 AAGAAGAAGA AGAAGAAGAAGAAGAAGAAG 262 <210> 8 <211> 285 <212> DNA <213> Zebrafish (Danio rerio) <400> 8 atttcccact cagcgtgtgt gtcagtgtgt gtctgtcata cttgcaatgg acagatggac 60 agcacagaca aatggataga tagatagata gatagataga tagatagata gatagataga 120 tagatagata gatagataga tagatagata gatgaaaata ggtaccaaca ttgtctgatt 180 cactccaaat gtctacactg aacacaattc cacataagct tgagaccatt gcagggtcac 240 tgtcacggct caacgtcaaa tgtcaaatga ctgaggagcc acagc 285

Claims

1. An SSR marker for oblique-banded grouper, characterized in that, Including ECDSSR-01, ECDSSR-02, ECDSSR-03, ECDSSR-04, ECDSSR-05, ECDSSR-06, ECDSSR-07 and ECDSSR-08: The nucleotide sequence of ECDSSR-01 is shown in SEQ ID NO.1; The nucleotide sequence of ECDSSR-02 is shown in SEQ ID NO.2; The nucleotide sequence of ECDSSR-03 is shown in SEQ ID NO.3; The nucleotide sequence of ECDSSR-04 is shown in SEQ ID NO.4; The nucleotide sequence of ECDSSR-05 is shown in SEQ ID NO.5; The nucleotide sequence of ECDSSR-06 is shown in SEQ ID NO.6; The nucleotide sequence of the ECDSSR-07 is shown in SEQ ID NO.7; The nucleotide sequence of ECDSSR-08 is shown in SEQ ID NO.

8.

2. A detection primer for the SSR marker as described in claim 1, for assessing the genetic diversity of a grouper population, characterized in that, The detection primers include: For ECDSSR-01 site: ECDSSR-01-F: 5'-GTCCTCTGCTTTGGGCTGAA-3'; ECDSSR-01-R: 5'-GAGTCACTGCACACTGACGA-3'; For the ECDSSR-02 site: ECDSSR-02-F: 5'-CCGTTGGCTGACTTTGAGC-3'; ECDSSR-02-R: 5'-CTGGAAGGCCTGTTCAGTCA-3'; For the ECDSSR-03 site: ECDSSR-03-F: 5'-TACTGCAGCACCACAGACTG-3'; ECDSSR-03-R: 5'- TGAACAGGCTGGTCTGCTTT -3'; For ECDSSR-04 site: ECDSSR-04-F: 5'- ACGCCATGCATGTCCATGTA -3'; ECDSSR-04-R: 5'- AGAGCCGCTTGTTCAAGAGG -3'; Targeting the ECDSSR-05 site: ECDSSR-05-F: 5'- AGGGTCTGCCTCTCCATCAT -3'; ECDSSR-05-R: 5'-AGACAGGGATCACTGCAAGC-3'; Targeting the ECDSSR-06 site: ECDSSR-06-F: 5'- GGGACAGGTGAGCAGATCAG -3'; ECDSSR-06-R: 5'- CCCATGAGGTGTCACTAGTGG -3'; For ECDSSR-07 site: ECDSSR-07-F: 5'- ATCCTTTATGCCAGGGCTGC -3'; ECDSSR-07-R: 5'- GCTCTTTGTCTGCCAACAGC -3'; For ECDSSR-08 site: ECDSSR-08-F: 5'- GCTGTGGCTCCTCAGTCATT -3'; ECDSSR-08-R: 5'-ATTTCCACTCAGCGTGTGT-3'.

3. The detection primer according to claim 2, characterized in that, The forward primer of the detection primer is labeled with a fluorescent group at its 5' end.

4. The detection primer according to claim 3, characterized in that, The fluorescent group is a FAM fluorescent group.

5. The application of the reagent for detecting the SSR marker described in claim 1 in the preparation of products for population genetic diversity analysis and population identification of *Sinocyclocheilus fasciatus*.

6. The use of the reagent for detecting the SSR marker as described in claim 1 or the detection primer as described in claim 2 in the preparation of a kit for analyzing the genetic diversity of groups of grouper and identifying populations.

7. A kit for analyzing the genetic diversity and identifying populations of *Scutellaria barbata*, characterized in that, The kit contains the detection primers as described in claim 2.

8. A method for assessing the genetic diversity of a grouper population, characterized in that, Includes the following steps: (1) Collect population samples of oblique-banded grouper and extract individual DNA from oblique-banded grouper; (2) Using the genomic DNA extracted in step (1) as a template, perform PCR amplification using the detection primers described in claim 2; (3) Genotyping the PCR products amplified in step (2); (4) Perform genetic diversity analysis on the typing results obtained in step (3).

9. The method according to claim 8, characterized in that, The PCR amplification described herein, in a 25 μL reaction system, comprises: Mg-free... 2+ Prepare 2.5 mL of 10×PCR buffer, 2.0 mL of 25 mM MgCl2, 0.5 mL of 10 mM dNTP, 1 U of high-fidelity PCR enzyme, 0.5 mL of 10 mM forward primer, 0.5 mL of 10 mM reverse primer, and 12.5 ng of DNA template. The remainder is made up to 25 mL with sterile double-distilled water. The reaction procedure is as follows: pre-denaturation at 95ºC for 5 minutes; denaturation at 95ºC for 30 seconds, annealing at 60ºC for 30 seconds, extension at 72ºC for 30 seconds, for a total of 35 cycles; extension at 72ºC for another 6 minutes.

10. The application of the SSR marker of claim 1, the detection primer of claim 2, the kit of claim 7, or the method of claim 8 in the genetic diversity analysis and geographical population identification of cultured grouper.

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