Microsatellite markers and their applications in brown-spotted grouper, clear-water grouper and their hybrid offspring, the shad hybrid grouper.

Abstract

This application belongs to the field of bioassay technology, and particularly relates to microsatellite markers and their applications for brown-spotted grouper, freshwater grouper, and their hybrid offspring, the 'Shanhu' hybrid grouper. This application provides SSR molecular markers for brown-spotted grouper, freshwater grouper, and their hybrid offspring, the 'Shanhu' hybrid grouper, including the following molecular markers: ad30, ae26, ab42, ad19, ad25, ae27, and ac34; the nucleotide sequences of the molecular markers are shown in SEQ ID NO.1 to SEQ ID NO.7, and the primer pairs for amplifying these molecular markers include the nucleotide sequences shown in SEQ ID NO.8 to SEQ ID NO.21. This application provides microsatellite markers for brown-spotted grouper, freshwater grouper, and their hybrid offspring, the 'Shanhu' hybrid grouper, and their applications, providing seven SSR molecular marker combinations that can be used for population genetic diversity analysis and identification of the three grouper species.

Description

Technical Field

[0001] This application belongs to the field of biological identification technology, and in particular relates to microsatellite markers and their applications for brown-spotted grouper, clear-water grouper and their hybrid offspring, the shad hybrid grouper. Background Technology

[0002] A hybridization breeding experiment was conducted using the brown-spotted grouper (commonly known as the tiger grouper) (Epinephelus fuscoguttatus) as the female parent and the clear-water grouper (commonly known as the cedar grouper) (E. polyphekadion) as the male parent, successfully obtaining the first-generation hybrid tiger-cedar grouper. Because the brown-spotted grouper and the clear-water grouper are similar in appearance, their hybrid offspring are difficult to distinguish from their parents. Therefore, an effective method or tool for identifying the brown-spotted grouper, the clear-water grouper, and their hybrid offspring, the tiger-cedar grouper, is urgently needed. Summary of the Invention

[0003] In view of this, this application provides microsatellite markers for brown-spotted grouper, clear-water grouper and their hybrid offspring, the shad hybrid grouper, and their applications, and provides seven SSR molecular marker combinations, which can be used for population genetic diversity analysis and identification of the three grouper species (brown-spotted grouper, clear-water grouper and their hybrid offspring, the shad hybrid grouper).

[0004] The first aspect of this application provides SSR molecular markers for brown-spotted grouper, clear-water grouper and their hybrid offspring, the shad hybrid grouper, including the following molecular markers: ad30, ae26, ab42, ad19, ad25, ae27 and ac34;

[0005] The nucleotide sequence of the ad30 molecular marker is shown in SEQ ID NO.1;

[0006] The nucleotide sequence of the ae26 molecular marker is shown in SEQ ID NO.2;

[0007] The nucleotide sequence of the ab42 molecular marker is shown in SEQ ID NO.3;

[0008] The nucleotide sequence of the ad19 molecular marker is shown in SEQ ID NO.4;

[0009] The nucleotide sequence of the ad25 molecular marker is shown in SEQ ID NO.5;

[0010] The nucleotide sequence of the ae27 molecular marker is shown in SEQ ID NO. 6;

[0011] The nucleotide sequence of the ac34 molecular marker is shown in SEQ ID NO.7.

[0012] Specifically, SEQ ID NO:

[0013] >ad30

[0014] TTGGCCGGGGAGGTCAGGAGGTGATGATGATGATGATGATGATGATGATGTTATCCCCCAGGGGACAGGTGAGATGCTACAGCTTGCTGTATGTTAGTTGCAAGCAGAAGA。

[0015] SEQ ID NO.2:

[0016] >ae26

[0017] CGTCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCTGTGATGGTGCTGTCTCTGTCCGTGGTGCTGAAGCATACACCAATCCCCATGCAGGGAAGAGCCCACTGAAAAAACACTGCTATGTCTGATGCTATGTCTGA。

[0018] SEQ ID NO.3:

[0019] >ab42

[0020] GGGGCTGCATGGGCTGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGCAGGCAGGCACAACGCCTTGGCGGCGGCGCGAGGAGGAGGGTTCACTGCGTTTGTGTTTCACCCCCCCCTTCCAGG。

[0021] SEQ ID NO.4:

[0022] >ad19

[0023] AGCGGACTGGTTGCTGGCGGGCAGGTGCGGCTCACAACAACAACAACAACAACAACAACAACAACAGCTGTATTTTTAGTATTTGATGTTGTTTACGTGGTTATTCTGGTCCATAAAAACCATAAAAA。

[0024] SEQ ID NO.5:

[0025] >ad25

[0026] AGGGCCCCTTATAATAATAATGATAATAATAATAATAATAATAATAATAATACATTCTATTTGTATGGCCGTTTCAAAACACTCAAATACACTTTATAAAAACAGAAAAAACAAGATAAAAACAACATAAGAGCAGCAAAACA.

[0027] SEQ ID NO.6:

[0028] >ad27

[0029] CGGACATTTTTCTTTTGGTCATGGTGAAATTAGAAATAAATGGTTTAGCCTCTCCTCCTCCTCCTCCTCCTCCTCATGACTGATAATAGAATGTATAGCTCCTCCAGGGTCATAA.

[0030] SEQ ID NO.7:

[0031] >ac34

[0032] CCCTATTTGATATTGATTGATATTGATTAATATGACATGAGGTAATATGATGATGATGATGATGATGATGATGATGATGATGTTTCTCATCCTCATCTTCCCCCTGCCTCAGACTCATCT.

[0033] In another embodiment, the microsatellite repeat sequence of the ad30 molecular marker is (TGA)9;

[0034] The microsatellite repeat sequence of the ae26 molecular marker is (GCA)9;

[0035] The microsatellite repeat sequence of the ab42 molecular marker is (GAG)10;

[0036] The microsatellite repeat sequence of the ad19 molecular marker is (ACA)10;

[0037] The microsatellite repeat sequence of the ad25 molecular marker is (TAA)9;

[0038] The microsatellite repeat sequence of the ae27 molecular marker is (CTC)9;

[0039] The microsatellite repeat sequence of the ac34 molecular marker is (ATG)11.

[0040] The second aspect of this application provides amplification primers for SSR molecular markers of brown-spotted grouper, clear-water grouper and their hybrid offspring, the shad hybrid grouper.

[0041] In another embodiment, it includes:

[0042] Primer pairs for amplifying the ad30 molecular marker include upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO. 8-9;

[0043] Primer pairs for amplifying the ae26 molecular marker include upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO. 10-11;

[0044] Primer pairs for amplifying the ab42 molecular marker include upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO. 12-13;

[0045] Primer pairs for amplifying the ad19 molecular marker include upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO. 14-15;

[0046] Primer pairs for amplifying the ad25 molecular marker include upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO. 16-17;

[0047] Primer pairs for amplifying the ae27 molecular marker include upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO. 18-19;

[0048] The primer pair for amplifying the ac34 molecular marker includes upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO. 20-21.

[0049] In another embodiment, the 5' end of the upstream primer of the amplification primer is labeled with a fluorescent group; the fluorescent group is ROX, FAM or HEX.

[0050] A third aspect of this application provides a kit for variety identification of brown-spotted grouper, clear-water grouper and their hybrid offspring, the shad tiger hybrid grouper, including the aforementioned amplification primers.

[0051] The fourth aspect of this application discloses the application of the SSR molecular marker, the amplification primer, or the variety identification kit in grouper genetic diversity analysis, grouper variety identification, and / or molecular marker-assisted breeding.

[0052] The fifth aspect of this application provides a method for analyzing the genetic diversity of grouper, including:

[0053] The SSR molecular marker and the amplification primers were used to amplify the grouper sample to be tested, and the electrophoresis detection was performed to obtain amplification products of different lengths and obtain genotype data.

[0054] The genotype data reading standard is as follows: for the same set of primers to amplify different grouper test samples, the largest band in the electrophoresis detection results is recorded as A, followed by B, and so on. Bands that are unclear or missing are recorded as 0. The genetic information of each set of primers is calculated, including the number of alleles, allele frequency, heterozygosity, expected heterozygosity, and polymorphism information content.

[0055] The genetic diversity results of the grouper samples to be tested were obtained based on the genetic information.

[0056] The sixth aspect of this application provides a method for identifying brown-spotted grouper, clear-water grouper, and their hybrid offspring, the shad-like hybrid grouper, characterized by comprising:

[0057] Using the SSR molecular marker and the amplification primers, PCR amplification was performed on brown-spotted grouper, clear-water grouper, and their hybrid offspring, the tiger grouper. Electrophoresis was used to detect amplification products of different lengths, and genotype data was obtained. The genotype data reading standard was as follows: for the same set of primers amplifying different groupers, the largest band in the electrophoresis detection results was recorded as A, followed by B, and so on. Unclear or missing bands were recorded as 0. The allele frequency table of standard grouper was calculated.

[0058] The SSR molecular marker and the amplification primers were used to perform PCR amplification on the grouper test samples, and the electrophoresis was used to detect the amplification products of different lengths, thereby obtaining the genotype data of the grouper test samples.

[0059] The genotype data of the grouper sample to be tested are compared with the allele frequency table of the standard grouper to determine the species of the grouper sample to be tested.

[0060] Specifically, conventional comparison methods are used to compare the genotype data of the grouper sample to be tested with the allele frequency table of the standard grouper to determine the species of the grouper sample to be tested.

[0061] In another embodiment, the PCR amplification program was set as follows: 95°C pre-denaturation for 5 min; 95°C denaturation, 59-60°C annealing, and 72°C extension for 30 sec each, for a total of 38 cycles; and finally 72°C extension for 10 min.

[0062] Currently, a hybridization breeding experiment has been conducted using the brown-spotted grouper (commonly known as the tiger grouper) (Epinephelus fuscoguttatus) as the maternal parent and the clear-water grouper (commonly known as the cedar grouper) (E. polyphekadion) as the paternal parent, successfully obtaining the F1 hybrid tiger-cedar grouper. Because the F1 hybrid tiger-cedar grouper is morphologically similar to both the maternal and paternal parents, a reliable and rapid tool or method for analyzing the genetic diversity and identifying varieties of these three species is needed. This application provides seven SSR molecular markers, including: ad30, ae26, ab42, ad19, ad25, ae27, and ac34. Primers for amplifying these SSR molecular markers can be used in the genetic diversity analysis and variety identification of these three populations, and can be applied in aquaculture production and sales. Attached Figure Description

[0063] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0064] Figure 1 The cluster diagram of the genetic distance between populations of brown-spotted grouper, clear-water grouper and their hybrid offspring, the shad hybrid grouper, calculated based on the amplification results of SSR molecular marker-based amplification primers provided in the embodiments of this application, was drawn using UPGMA software. Detailed Implementation

[0065] This application provides microsatellite markers for brown-spotted grouper, clear-water grouper and their hybrid offspring, the shad hybrid grouper, and their applications. It also provides an SSR molecular marker and its primer pair that can simultaneously or separately determine the genetic diversity analysis of grouper, identify grouper varieties and / or marker-assisted breeding.

[0066] The technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0067] In the following examples, all raw materials or reagents used are commercially available or self-made.

[0068] The microsatellite molecular markers in this application, also known as short tandem repeats (STRs) or simple sequence repeats (SSRs), are simple repetitive sequences uniformly distributed throughout the genomes of eukaryotes. They consist of tandem repeat fragments of 2–6 nucleotides. Due to the high variability in the number of repeats per repeat unit among individuals and their abundance, microsatellite markers have a wide range of applications. Microsatellite loci are typically amplified by PCR, and the amplification products are analyzed by electrophoresis, with alleles separated by size for detection.

[0069] The experimental subjects used in the following examples were: brown-spotted grouper (tiger grouper), clear-water grouper (cedar grouper), and hybrid offspring of brown-spotted grouper and clear-water grouper (cedar-tiger hybrid grouper). Information such as the genetic diversity of the population can be calculated based on the fragment information amplified by microsatellite marker primers.

[0070] The molecular markers and amplification primers used in the following examples for brown-spotted grouper, clear-water grouper and their hybrid offspring, shad hybrid grouper, include: ad30 molecular marker, ae26 molecular marker, ab42 molecular marker, ad19 molecular marker, ad25 molecular marker, ae27 molecular marker and ac34 molecular marker;

[0071] The nucleotide sequence of the ad30 molecular marker is shown in SEQ ID NO.1. The primer pair for amplifying the ad30 molecular marker includes upstream and downstream primers with nucleotide sequences shown in SEQ ID NO.8-9.

[0072] The nucleotide sequence of the ae26 molecular marker is shown in SEQ ID NO.2. The primer pair for amplifying the ae26 molecular marker includes upstream and downstream primers with nucleotide sequences shown in SEQ ID NO.10-11.

[0073] The nucleotide sequence of the ab42 molecular marker is shown in SEQ ID NO.3. The primer pair for amplifying the ab42 molecular marker includes upstream and downstream primers with nucleotide sequences shown in SEQ ID NO.12-13.

[0074] The nucleotide sequence of the ad19 molecular marker is shown in SEQ ID NO.4. The primer pair for amplifying the ad19 molecular marker includes upstream and downstream primers with nucleotide sequences shown in SEQ ID NO.14-15.

[0075] The nucleotide sequence of the ad25 molecular marker is shown in SEQ ID NO.5. The primer pair for amplifying the ad25 molecular marker includes upstream and downstream primers with nucleotide sequences shown in SEQ ID NO.16-17.

[0076] The nucleotide sequence of the ae27 molecular marker is shown in SEQ ID NO.6. The primer pair for amplifying the ae27 molecular marker includes upstream and downstream primers with nucleotide sequences shown in SEQ ID NO.18-19.

[0077] The nucleotide sequence of the ac34 molecular marker is shown in SEQ ID NO.7. The primer pair for amplifying the ac34 molecular marker includes upstream and downstream primers with nucleotide sequences shown in SEQ ID NO.20-21.

[0078] The relevant information of the molecular markers and amplification primers of this application is shown in Table 1.

[0079] Table 1

[0080]

[0081]

[0082] Note: F indicates the forward primer, and R indicates the reverse primer.

[0083] Example 1

[0084] This application provides a screening process for SSR molecular markers in brown-spotted grouper, clear-water grouper, and their hybrid offspring, the shad hybrid grouper. The specific steps include:

[0085] 1. The microsatellite molecular marker screening process utilizes ddRAD-seq (Double digest restriction-site associated DNA sequencing) technology. An extracted brown-spotted grouper DNA sample was sent to Beijing Novogene Technology Co., Ltd. to construct a RAD library. The library was constructed using the 6-base enzyme EcoR1, and corresponding adapters were added to both ends of the fragments. After amplification, a sequencing library was constructed. High-throughput sequencing was performed using a Paired-End 100 strategy on an Illumina HiSeq2000. The sequencing data was clustered by RAD tags to generate assembled apire-end reads (read 2). Contig detection and assembly yielded restriction-associated contigs of approximately 300 bp in length, used for SSR marker development. Based on the sequencing results, SSR markers were initially screened using MISA software. MISA parameters were set to at least 6 dinucleotide repeats, 5 trinucleotide repeats, 5 tetranucleotide repeats, 5 pentanucleotide repeats, and 5 hexanucleotide repeats, with an interval of at least 100 bp between two SSRs. Then, initial screening was performed, randomly selecting 80 dinucleotide repeats with 10 to 25 repeats and 6 to 20 trinucleotide repeats for primer synthesis. PCR amplification was then performed, with parallel controls. Each pair of microsatellite primers was amplified using 8 DNA templates. The total PCR reaction volume was 10 μL, including 5 μL of 2×Taq PCR StarMix, 3.2 μL of ddH2O, 0.4 μL each of the 3' and 5' microsatellite primers, and 1 μL of DNA template. The PCR program was set as follows: 95℃ pre-denaturation for 5 min; followed by 10 cycles of 95℃, 68℃, and 72℃ for 30 sec each, decreasing the temperature by 1℃ per cycle; then 25 cycles of 95℃, 58℃, and 72℃ for 30 sec each; finally, an extension at 72℃ for 10 min. PCR products were detected by 1.5% agarose gel electrophoresis at 120V for 16 min, and the bands were observed. Microsatellite markers with a single bright band were retained during agarose gel electrophoresis to confirm primer usability. Then, polyacrylamide gel electrophoresis (PAGE) was performed. PCR products were electrophoresed on an 8% non-denaturing polyacrylamide gel, with 3 μL of PCR product and 3 μL of DNA marker (10 bp DNA ladder) loaded per lane. Electrophoresis buffer was 0.5×TBE, and electrophoresis was performed at 600V for 1 h. After electrophoresis, silver nitrate staining was performed. Finally, the gel was laid flat and photographed for analysis, followed by screening for microsatellite loci polymorphism. Based on the electrophoresis results, microsatellite loci with 3 or more alleles and no non-specific amplification were selected, and then population amplification was performed to obtain multiple microsatellite loci.

[0086] 2. The 5' end of the forward primer for each microsatellite locus after screening was labeled with a fluorescent dye. The PCR program was set as follows: 95℃ pre-denaturation for 5 min; 38 cycles of 95℃, 55℃, and 72℃ for 30 sec each; and a final extension at 72℃ for 10 min.

[0087] PCR products were subjected to capillary electrophoresis for short tandem repeat (STR) genotyping using an ABI 3730 Analyzer. The results were then analyzed using Genemapper 4.0 software, with LIZ500 as the internal standard. This method can detect FAM, HEX, and ROX fluorescence. The peak values ​​were used to determine the degree of polymorphism of microsatellite loci in the three populations, allowing for more precise screening of microsatellite loci.

[0088] 3. After the above screening, the embodiments of this application obtained 7 microsatellite molecular loci with high specificity and high polymorphism in three populations: brown spot grouper, clear water grouper, and spruce tiger hybrid grouper (see molecular markers, Table 2).

[0089] Table 2 shows the primer sequences and annealing temperatures for the seven SSR sites provided in this application.

[0090]

[0091]

[0092] Note: F indicates the forward primer, and R indicates the reverse primer.

[0093] Example 2

[0094] This application's embodiments calculate the SSR loci and allele frequencies in brown-spotted grouper, clear-water grouper, and spruce-tiger hybrid grouper. Specific steps include:

[0095] Thirty individuals from each population (brown-spotted grouper, clear-water grouper, and shad hybrid grouper) were subjected to capillary electrophoresis STR typing. The accurate PCR product lengths of microsatellite molecular markers for brown-spotted grouper, clear-water grouper, and shad hybrid grouper were obtained based on the peak values. The genetic information of each primer pair could be calculated, including the number of alleles, allele frequency (Table 3), heterozygosity, expected heterozygosity, and polymorphism information content.

[0096] The above calculation method includes: converting the data into Cervus format using the Excel plugin Genalex; inputting the converted file into Cervus software; clicking "analysis" to bring up the input interface; entering the file format in "genotype"; entering the output path and output file name in "summary output file"; clicking "ID in column" to input the column number of the sample; clicking "first allele in column" to input the column number from which the first allele starts; and clicking "numberofloci" to input the number of loci in the file. After clicking "OK," the output results can calculate the allele number (A), allele frequency, and use Cervus software to calculate observed heterozygosity (Ho), expected heterozygosity (He), and polymorphic information content (PIC).

[0097] Table 3 shows the statistics of 7 SSR loci and allele frequencies in brown-spotted grouper, clear-water grouper, and hybrid offspring. Table 4 shows the statistics of genetic variation parameters in brown-spotted grouper, clear-water grouper, and hybrid offspring.

[0098] Table 3 shows the loci / alleles, which represent the nucleic acid lengths (bp) of each SSR molecular marker. In Table 3, ad30 indicates that: when PCR was performed on brown-spotted grouper using primers that amplified the ad30 molecular marker, 139bp, 142bp, 145bp, 148bp, and 151bp amplification products were obtained. When PCR was performed on freshwater grouper using primers that amplified the ad30 molecular marker, 136bp, 139bp, and 148bp amplification products were obtained.

[0099] Table 3. Statistics on the SSR loci and allele frequencies of seven SSR loci in brown-spotted grouper, clear-water grouper, and hybrid offspring.

[0100]

[0101]

[0102] Note: In the table above, " / " indicates no amplified band.

[0103] Table 4. Genetic variation parameters of brown-spotted grouper, clear-water grouper, and hybrid offspring.

[0104]

[0105]

[0106]

[0107] Example 3

[0108] This application provides a method for identifying brown-spotted grouper, clear-water grouper, and their hybrid offspring, the shad tiger hybrid grouper, specifically including:

[0109] 1. Using the amplification primers (SEQ ID NO. 8~SEQ ID NO. 21) of the above-mentioned SSR molecular markers, PCR amplification was performed on brown-spotted grouper, clear-water grouper and their hybrid offspring, the shad hybrid grouper, respectively. Electrophoresis was performed to obtain amplification products of different lengths and genotype data. The genotype data reading standard was as follows: for the same set of primers amplifying different groupers, the largest band in the electrophoresis detection results was recorded as A, followed by B, and so on. The unclear band or missing band was recorded as 0. The allele frequency table of standard grouper was calculated according to the method described in the literature (i.e., Table 3).

[0110] 2. When identifying an individual of an unknown grouper species, the process includes: ① Extracting DNA from the individual of the unknown grouper species and performing PCR amplification using the seven primer pairs (SEQ ID NO. 8~SEQ ID NO. 21) mentioned above. Based on the amplification results, compare them with the seven microsatellite loci and allele frequencies in Table 3. If the amplification result of the unknown grouper species corresponds to an individual of a certain species, or if a specific locus amplified by a certain primer is found in a certain species, the species to which the individual belongs can be determined. For example, if loci 145 or 151 appear in the amplification result of primer ad30, the species can be identified as brown-spotted grouper (tiger grouper). If no specific locus appears in the amplification results of any of the seven primer pairs, a comprehensive determination is required. For example, if the amplification result of primer ad30 for the unknown grouper species is 136 and the amplification result of primer ae26 is 150, the species to which this individual belongs can be identified as clear-water grouper (cedar grouper).

[0111] Example 4

[0112] This application embodiment calculates the interpopulation genetic distance of the above-mentioned brown-spotted grouper, clear-water grouper, and their hybrid offspring, the spruce tiger hybrid grouper, specifically including:

[0113] Based on the genetic distances between populations calculated from the electrophoresis results, the genetic distance between the hybrid offspring and the maternal brown-spotted grouper is 0.268, and the genetic distance between the hybrid offspring and the paternal clear-water grouper is 0.269.

[0114] Example 5

[0115] The data was converted to Cervus format using the Excel plugin Genalex. The converted file was then input into Cervus software, which output data in a suitable format. This data was then run in POPGENE32, and a phylogenetic tree based on Nei's genetic distance was generated. The tree was opened with Mega and a clustering diagram of genetic distances among the populations of Brown Spotted Grouper, Clearwater Grouper, and their hybrid offspring, the Spotted Tiger Grouper, was obtained using UPGMA. Figure 1 It can be seen that the hybrid offspring and brown-spotted grouper aggregated first, and then the two aggregated with clear-water grouper, indicating that the seven pairs of microsatellite primers can effectively distinguish the three populations.

[0116] In summary, the ad30, ae26, ab42, ad19, ad25, ae27, and ac34 molecular markers provided in this application can be simultaneously applied to the genetic diversity analysis, grouper variety identification, and / or marker-assisted breeding of three species.

[0117] The above description is only a preferred embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. Primers for amplifying SSR molecular markers in brown-spotted grouper, clear-water grouper, and their hybrid offspring, the juvenile tiger grouper, characterized in that... The SSR molecular markers include ad30, ae26, ab42, ad19, ad25, ae27, and ac34; The nucleotide sequence of the ad30 molecular marker is shown in SEQ ID NO.1; The nucleotide sequence of the ae26 molecular marker is shown in SEQ ID NO.2; The nucleotide sequence of the ab42 molecular marker is shown in SEQ ID NO.3; The nucleotide sequence of the ad19 molecular marker is shown in SEQ ID NO.4; The nucleotide sequence of the ad25 molecular marker is shown in SEQ ID NO.5; The nucleotide sequence of the ae27 molecular marker is shown in SEQ ID NO. 6; The nucleotide sequence of the ac34 molecular marker is shown in SEQ ID NO.7; The microsatellite repeat sequence of the ad30 molecular marker is (TGA)9; The microsatellite repeat sequence of the ae26 molecular marker is (GCA)9; The microsatellite repeat sequence of the ab42 molecular marker is (GAG)10; The microsatellite repeat sequence of the ad19 molecular marker is (ACA)10; The microsatellite repeat sequence of the ad25 molecular marker is (TAA)9; The microsatellite repeat sequence of the ae27 molecular marker is (CTC)9; The microsatellite repeat sequence of the ac34 molecular marker is (ATG)11; The amplification primers include: Primer pairs for amplifying the ad30 molecular marker include upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO. 8-9; Primer pairs for amplifying the ae26 molecular marker include upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO. 10-11; Primer pairs for amplifying the ab42 molecular marker include upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO. 12-13; Primer pairs for amplifying the ad19 molecular marker include upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO. 14-15; Primer pairs for amplifying the ad25 molecular marker include upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO.16-17; Primer pairs for amplifying the ae27 molecular marker include upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO. 18-19; The primer pair for amplifying the ac34 molecular marker includes upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO. 20-21.

2. The SSR molecular marker amplification primers according to claim 1, characterized in that, The 5' end of the upstream primer of the amplification primer is labeled with a fluorescent group; the fluorescent group is ROX, FAM or HEX.

3. A kit for variety identification of brown-spotted grouper, clear-water grouper and their hybrid offspring, the shad tiger hybrid grouper, characterized in that, Amplification primers for the SSR molecular markers as described in any one of claims 1 to 2.

4. The application of the SSR molecular marker amplification primer according to any one of claims 1 to 2 in the genetic diversity analysis of brown-spotted grouper, clear-water grouper and their hybrid offspring, the juvenile tiger hybrid.

5. The application of the variety identification kit according to claim 3 in the identification of brown-spotted grouper, clear-water grouper and their hybrid offspring, shad tiger hybrid grouper.

6. A method for analyzing the genetic diversity of brown-spotted grouper, clear-water grouper, and their hybrid offspring, the juvenile tiger grouper, characterized in that... include: Using the amplification primers described in any one of claims 1 to 2, the samples of brown-spotted grouper, clear-water grouper and their hybrid offspring, shad hybrid grouper, were amplified and detected by electrophoresis to obtain amplification products of different lengths and obtain genotype data. The genotype data reading standard is as follows: for the same set of primers to amplify different grouper test samples, the largest band in the electrophoresis detection results is recorded as A, followed by B, and so on. Bands that are unclear or missing are recorded as 0. The genetic information of each set of primers is calculated, including the number of alleles, allele frequency, heterozygosity, expected heterozygosity, and polymorphism information content. Genetic diversity results of the tested samples of brown-spotted grouper, clear-water grouper and their hybrid offspring, spruce tiger hybrid grouper, were obtained based on the genetic information.

7. A method for identifying brown-spotted grouper, clear-water grouper, and their hybrid offspring, the juvenile tiger grouper, characterized in that... include: Using the amplification primers described in any one of claims 1-2, PCR amplification was performed on brown-spotted grouper, clear-water grouper, and their hybrid offspring, the juvenile grouper, followed by electrophoresis detection to obtain amplification products of different lengths and genotype data. The genotype data reading standard was as follows: for amplification of different groupers using the same set of primers, the largest band in the electrophoresis detection results was recorded as A, followed by B, and so on, with unclear or missing bands recorded as 0. The allele frequency table of standard grouper was calculated. Using the amplification primers described in any one of claims 1 to 2, PCR amplification was performed on the grouper test sample, followed by electrophoresis detection to obtain amplification products of different lengths, thereby obtaining the genotype data of the grouper test sample. The genotype data of the grouper sample to be tested are compared with the allele frequency table of the standard grouper to determine the species of the grouper sample to be tested.

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