Multiplex pcr fluorescent labeled primer set for epinephelus lanceolatus, application and kit and method

Abstract

The application belongs to the technical field of microsatellite markers, and particularly relates to a kind of multiplex PCR SSR fluorescently labeled primer set of Epinephelus lanceolatus, application and kit and method.The primer set of the application is specific primer pair and universal primer pair;Among them, the specific primer pair is G1 group and / or G2 group, the G1 group is the primer pair El-01-El-05 shown in the base of SEQ ID NO:1-10, and the G2 group is the primer pair El-06-El-10 shown in the base of SEQ ID NO:11-20.The primer of the application is high in polymorphism, stable in product, and accurate and reliable in result;The method is applied in monitoring genetic diversity and germplasm resource protection of Epinephelus lanceolatus.

Description

Technical Field

[0001] This invention belongs to the field of microsatellite labeling technology, specifically relating to an SSR fluorescent labeling primer set, application, kit, and method for multiplex PCR of saddleback grouper. Background Technology

[0002] In the grouper aquaculture industry system, the saddle-tailed grouper ( Epinephelus lanceolatus Not only do they possess high economic value, but their growth advantages and superior traits have also long served as "key parents" in hybridization breeding. For example, they are used as male parents with female brown-spotted grouper (… Epinephelus fuscoguttatus Hybridization produces the pearl grouper. However, the current grouper industry chain faces pressure regarding parent stock and germplasm management: closed-group breeding may accumulate inbreeding risks and induce phenotypic degeneration and decreased disease resistance; while large-scale application of hybrids, cross-regional distribution of seedlings, and potential escape from aquaculture may lead to hybridization and genetic contamination, making reliable identification and traceability difficult to achieve solely through morphology (Kuo et al. 2014). Therefore, accurate and high-throughput molecular detection methods are urgently needed for parent purity control, hybrid offspring identification, seedling traceability, and monitoring of potential genetic contamination.

[0003] Microsatellites (SSRs) are short tandem repeat sequences (1–6 bp) widely distributed in the genome. They possess advantages such as high polymorphism, codominance, good reproducibility, and rich information content, making them suitable for individual genetic fingerprinting, parentage testing, family management, and population genetic diversity analysis, without being affected by phenotypic traits such as body color and size. Current identification methods for saddleback grouper still rely to some extent on body color, markings, and morphological characteristics. However, these methods have insufficient distinguishing ability for closely related species, hybrid offspring, and batch seedlings. Furthermore, there is a lack of accurate identification of grouper and its hybrids in the market and aquaculture distribution channels. Therefore, it is necessary to establish an accurate, stable, and batch-suitable SSR typing method for saddleback grouper to meet the practical needs of its germplasm identification and genetic analysis.

[0004] However, although existing studies have reported on the development and paternity analysis of SSRs in *Gymnocypris saddleback*, the number of publicly disclosed loci is limited, and existing loci cannot be directly used to form a stable multiplex PCR system under uniform reaction conditions. Furthermore, current SSR detection methods often involve amplifying a small number of loci separately and genotyping them site-by-site. With larger sample sizes, this significantly increases the number of PCR reactions and genotyping steps, making the process cumbersome and prone to introducing pipetting errors and batch-to-batch variations, thus reducing detection efficiency and increasing overall costs. Therefore, it remains necessary to establish a multi-locus multiplex PCR-SSR detection / genotyping system suitable for *Gymnocypris saddleback* to achieve simultaneous amplification and rapid genotyping of multiple target loci, thereby meeting its application needs in germplasm identification, family management, and genetic analysis. Summary of the Invention

[0005] The purpose of this invention is to provide an SSR fluorescently labeled primer set, application, kit, and method for multiplex PCR of saddleback grouper.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A set of SSR fluorescently labeled primers for multiplex PCR of saddleback grouper, comprising specific primer pairs and universal primer pairs; wherein the specific primer pairs are G1 group and / or G2 group, wherein the G1 group consists of primer pairs E1-01-E1-05 as shown in SEQ ID NO: 1-10, and the G2 group consists of primer pairs E1-06-E1-10 as shown in SEQ ID NO: 11-20.

[0007] Each primer pair consists of a forward primer F and a reverse primer R; wherein: The base sequences of the forward and reverse primers for the primer pair El-01 are shown in SEQ ID NO: 1-2, respectively; The base sequences of the forward and reverse primers for the primer pair E1-02 are shown in SEQ ID NO: 3-4, respectively. The base sequences of the forward and reverse primers of the primer pair E1-03 are shown in SEQ ID NO: 5-6, respectively; The base sequences of the forward and reverse primers for the primer pair E1-04 are shown in SEQ ID NO: 7-8, respectively; The base sequences of the forward and reverse primers for the primer pair E1-05 are shown in SEQ ID NO: 9-10, respectively; The base sequences of the forward and reverse primers for the primer pair E1-06 are shown in SEQ ID NO: 11-12, respectively. The base sequences of the forward and reverse primers for the primer pair E1-07 are shown in SEQ ID NO: 13-14, respectively. The base sequences of the forward and reverse primers for the primer pair E1-08 are shown in SEQ ID NO: 15-16, respectively. The base sequences of the forward and reverse primers for the primer pair E1-09 are shown in SEQ ID NO: 17-18, respectively. The base sequences of the forward and reverse primers for the primer pair E1-10 are shown in SEQ ID NO: 19-20, respectively.

[0008] The universal primer pair is fluorescently labeled universal primers M13 and PQE-F; wherein the base sequence of M13 is shown in SEQ ID NO: 21, and the base sequence of PQE-F is shown in SEQ ID NO: 22.

[0009] The fluorescent labels used are 5-FAM, 5-HEX, VIC, NED, or PET, respectively.

[0010] The fluorescent label that works with the universal primer M13 is 5-FAM; The fluorescent label that works in conjunction with the universal primer PQE-F is 5-HEX.

[0011] A kit for multiplex PCR of saddleback grouper, the kit containing the SSR fluorescently labeled primer set for multiplex PCR of saddleback grouper.

[0012] Application of the SSR fluorescently labeled primer set for multiplex PCR of the saddle-banded grouper, and its application in germplasm resource evaluation and pedigree identification of the saddle-banded grouper. Alternatively, the SSR fluorescently labeled primer set for multiplex PCR of the saddle-banded grouper may be used to assess the genetic diversity of the saddle-banded grouper and to promote the development and protection of fishery resources.

[0013] An application of the kit described above, specifically its application in germplasm resource evaluation and pedigree identification of the saddle-banded grouper; Alternatively, the kit may be used to assess the genetic diversity of saddleback grouper and to promote the development and conservation of fishery resources.

[0014] A method for SSR typing of saddleback grouper: (1) Extract genomic DNA from the sample to be tested; (2) The genomic DNA in step (1) is amplified by multiplex PCR using the specific primer pair to obtain the amplification product; (3) Amplify specific bands with a length range of 250-520bp and a gradient. The sample to be tested contains saddle-banded grouper. At the same time, genotyping is performed based on the product. The genotyping information is used for the genetic diversity analysis of the saddle-banded grouper population.

[0015] The specific primer pair used in step (2) is the E1-01—E1-05 primer pair (i.e., G1 group). The total volume of the G1 group multiplex PCR amplification reaction system is 50 μL, including: 25 μL 2×Taq PCR Master Mix, 2 μL genomic DNA, 0.1 μL each of 10 μM forward primers, 0.4 μL each of 10 μM reverse primers, 2.4 μL of 10 μM fluorescently labeled universal primer M13, 2.4 μL of 10 μM fluorescently labeled universal primer PQE-F, 1.6 μL of 10 μM fluorescently labeled universal primer M13, 1.5 μL bovine serum albumin (BSA), and the remainder is made up to 50 μL with ultrapure water. In the multiplex PCR amplification reaction system, the volume ratio of the forward primer to the reverse primer in each primer pair is 1:4.

[0016] The specific primer pairs and fluorescently labeled universal primers for the amplification of the G1 group microsatellite loci are shown in Table 1: Table 1. Specific primer pairs and fluorescently labeled universal primers for G1 group microsatellite locus amplification The specific primer pair used in step (2) is the primer pair of E1-06—E1-10 (i.e., G2 group). The total volume of the G2 group multiplex PCR amplification reaction system is 50 μL, including: 25 μL 2×Taq PCR Master Mix, 2 μL genomic DNA, 0.1 μL each of 10 μM forward primers, 0.4 μL each of 10 μM reverse primers, 2.4 μL of 10 μM fluorescently labeled universal primer M13, 2.4 μL of 10 μM fluorescently labeled universal primer PQE-F, 1.6 μL of 10 μM fluorescently labeled universal primer M13, 1.5 μL bovine serum albumin (BSA), and the remainder is made up to 50 μL with ultrapure water. In the multiplex PCR amplification reaction system, the volume ratio of the forward primer to the reverse primer in each primer pair is 1:4; Table 2 shows the specific primer pairs and fluorescently labeled universal primers for G2 group microsatellite locus amplification. Table 2. Specific primer pairs and fluorescently labeled universal primers for microsatellite locus amplification in group G2. The configuration of the multiplex PCR system is determined based on the amplification performance of each primer pair and the interactions between primers. The specific steps are as follows: First, adjust and determine the optimal working concentration of each primer based on the preliminary amplification results (band clarity, specificity, and peak shape), and prepare crude buffer solutions for each primer. Then, premix each primer pair at a ratio of forward primer to reverse primer of 1:4 to obtain a pre-mixed solution for each primer pair. Next, mix the pre-mixed solutions of each primer pair according to the specified ratio to prepare the total multiplex PCR primer mixture. Finally, prepare the amplification reaction system all at once according to the number of samples to be tested, aliquot it into each reaction tube, and add template DNA for amplification. This premix-total-sample preparation process effectively reduces pipetting errors caused by adding primers one by one, saves reagents and time, improves experimental efficiency and repeatability, and is suitable for stable amplification of batch samples.

[0017] The amplification program used for the multiplex PCR amplification was as follows: 98 ℃ for 10 s, 57 ℃ for 40 s, 72 ℃ for 60 s, 35 cycles; 98 ℃ for 10 s, 53 ℃ for 40 s, 72 ℃ for 60 s, 15 cycles; and a final extension at 72 ℃ for 30 min.

[0018] The genotyping method employs capillary electrophoresis to genotype the amplification products on an ABI 3730XL gene analyzer.

[0019] Compared with the prior art, the present invention has the following advantages: (1) The microsatellite loci contained in this invention are 3 to 4 bases. Based on the genomic data of the saddle-banded grouper, they were obtained through "site-directed screening - single PCR pre-screening - multiplex system compatibility optimization". The primer amplification is stable, the typing is clear and the polymorphism is high, which improves the accuracy of genotype data. (2) This invention utilizes a combination of microsatellite markers, multiplex PCR and universal amplification primers, and uses 10 highly polymorphic microsatellite loci and designs specific primers based on these microsatellite loci to genotype saddle-banded grouper. When used for batch detection, it ensures the reliability of individual discrimination and genetic diversity assessment results. (3) In practical applications, the primer pairs of this invention can be used alone with group G1 to detect 5 sites; or with group G2 to detect 5 sites; or by combining groups G1 and G2 at once to detect 10 sites simultaneously. In practical applications in aquaculture, the appropriate number of primers can be selected for amplification based on the number of samples, and the universal primers can be reused. Compared with simple single-site detection, the cost is reduced and the experimental efficiency is higher. (4) The SSR typing method for saddle-banded grouper in this invention uses a reliable and effective combination of microsatellite primers and multiplex PCR technology to identify the specificity of saddle-banded grouper populations and perform typing. This method can be used for genetic diversity assessment, pedigree identification and fishery resource development and protection. Attached Figure Description

[0020] Figure 1 The images shown are agarose gel electrophoresis images from Example 2; a) shows 10 bands from left to right in the saddle-banded grouper, corresponding to sites E1-01, E1-02, E1-03, E1-04, E1-05, E1-06, E1-07, E1-08, E1-09, and E1-10, respectively; b) shows the gel electrophoresis images of the PCR amplification products of the 10 SSR sites of the saddle-banded grouper in Example 2 in brown-spotted grouper and pearl grouper.

[0021] Figure 2This is a detailed capillary electrophoresis image of sites El-01, El-02, El-03, El-04, and El-05 in group G1 of Example 3; where a is site El-01, b is site El-02, c is site El-03, and d is sites El-04 and El-05.

[0022] Figure 3 This is a detailed capillary electrophoresis image of sites El-06, El-07, El-08, El-09, and El-10 in group G2 of Example 3. In the image, a represents site El-06, b represents site El-07, c represents site El-08, and d represents sites El-09 and El-10. Detailed Implementation

[0023] The specific implementation of the present invention will be further illustrated below with examples.

[0024] This invention provides primer sets for multiplex PCR-SSR markers with repeating motifs of 3-4 bases in length; these primer sets enable fingerprint identification, germplasm identification, and population genetic diversity analysis of individual grouper. The method provides accurate identification results, high experimental efficiency, low cost, and sample saving, making it suitable for batch experiments.

[0025] Example 1 The SSR fluorescently labeled primers for multiplex PCR of *Gymnocypris saddleback* include 10 pairs of specific primers, namely primer pairs E1-01, E1-02, E1-03, E1-04, E1-05, E1-06, E1-07, E1-08, E1-09, and E1-10. Each primer pair includes one forward primer and one reverse primer. The base sequences of the 10 pairs of specific primers are shown in SEQ ID NO: 1–20. It also includes two fluorescently labeled universal primers, M13 and PQE-F. The fluorescent label associated with universal primer M13 is 5-FAM, and the fluorescent label associated with universal primer PQE-F is 5-HEX. The base sequences of the universal primers are shown in SEQ ID NO: 21–22. Specific sequences are shown in Tables 3 and 4 below. Table 3. Base sequences of 10 pairs of the above-mentioned specific primers Table 4. Two universal primers and fluorescent labels that work in conjunction with the universal primers. Download Saddleback Grouper from GenBank ( Epinephelus lanceolatusThe genome, submitted under submission number GCA_005281545.1, has a total length of 1086 Mb, a GC content of 41.26%, a scaffold N50 length of 46.2 Mb, and a contig N50 length of 119.9 kb. MISA software was used to mine SSR loci across the entire genome, prioritizing candidate SSR loci with 3–4 bp repeat units, clear repeat structures, and stable flanking sequences. Further screening was conducted based on their potential polymorphism and genotyping application requirements. On this basis, 45 candidate primer pairs were designed using primer design software, and singleton PCR was performed on 6 individuals of *Gymnocypris saddleback* to pre-screen them, eliminating non-specific amplification, unstable amplification, and loci without significant polymorphism. Finally, 10 SSR loci with similar Tm values, reasonable product length distribution, stable amplification, and good polymorphism were retained. Through a combination of bioinformatics screening and experimental verification, the amplification specificity of primers and the compatibility between primer combinations were evaluated. Two sets of multiplex SSR-PCR combinations were selected, denoted as G1 and G2, each containing 5 microsatellite loci, as shown in Tables 5 and 6 below. The microsatellite sequences were labeled with fluorescent universal primers and automatically genotyped using capillary electrophoresis.

[0026] Table 5. Primer pairs for G1 group microsatellite locus amplification Table 6. Primer pairs for G2 group microsatellite loci amplification Example 2 This embodiment provides proof of primer specificity for multiplex PCR of grouper with saddleback fins. Using the aforementioned 10 pairs of specific primers, multiple target fragments were amplified by PCR. The amplification products of different primers were separated by electrophoresis, and the separated bands were statistically analyzed. Specifically: (1) Extraction of genomic DNA from different fish species: Collected from *Spodoptera litura* and its closely related species *Spodoptera chinensis* (…). Epinephelus fuscoguttatus ) and hybrid pearl grouper ( ♀ E. fuscoguttatus × ♂ E. lanceolatus Genomic DNA was extracted from the fin tissue samples.

[0027] (2) Using the primer pairs obtained in the above examples, PCR amplification was performed. The PCR amplification system was as follows: the total reaction volume was 25 μL, including 12.5 μL of TaKaRa 2×Taq PCR Master Mix, 1 μL (10 μM) of each primer pair (forward and reverse) at appropriate concentrations, and an appropriate amount of template DNA (100 ng). The remainder was made up with sterile water. PCR amplification: the amplification program was 98℃ for 10 s, 57℃ for 40 s, 72℃ for 60 s, for 35 cycles, with a final extension at 72℃ for 30 min.

[0028] (3) Take 5 μL of the amplification product for agarose gel electrophoresis. The electrophoresis results are as follows: Figure 1 As shown. From Figure 1 As can be seen from Figure a, the primers corresponding to E1-01 to E1-10 all amplified bright, clearly defined target bands in *Gymnocypris saddle-shaped*, with no obvious impurities or primer dimers. However, no obvious amplification products were observed in *Gymnocypris purpurea* and *Gymnocypris pulcherrima* (see Figure a). Figure 1 (b) indicates that these primers have good specificity and amplification stability in grouper; at the same time, the amplification products 200-520 bp are all within the target range, with clear partitioning, and can be used for the construction of subsequent multiplex PCR systems.

[0029] Example 3 The following specific examples illustrate how the SSR fluorescently labeled primer set and SSR genotyping method obtained in the above examples are used to assess the genetic diversity of the saddleback grouper. (1) Extract DNA from saddle-shaped grouper: Fin rays from 30 individual saddle-shaped grouper were cut and immediately preserved in 95% ethanol. These samples are designated as P1-P30. Whole-genome DNA was extracted using a marine animal tissue genomic DNA extraction kit. For specific steps, please refer to the kit instructions. After DNA extraction, the concentration was detected using a UV spectrophotometer.

[0030] (2) Primer set The primers listed in Tables 5 and 6 above were used.

[0031] (3) The total volume of the multiplex PCR amplification reaction system is 50 μL, including: 25 μL 2×Taq PCR Master Mix, 2 uL genomic DNA, 0.1 μL each of 10 μM forward primers, 0.4 μL each of 10 μM reverse primers, 2.4 μL of 10 μM fluorescently labeled universal primer M13, 2.4 μL of 10 μM fluorescently labeled universal primer PQE-F, 1.6 μL of 10 uM fluorescently labeled universal primer M13, 1.5 μL bovine serum albumin (BSA), and the remainder is made up to 50 uL with ultrapure water.

[0032] PCR reaction program settings: 98℃ for 10s, 59℃ for 30s, 72℃ for 60s, 30 cycles; 98℃ for 10s, 53℃ for 30s, 72℃ for 60s, 15 cycles; final extension at 72℃ for 30min.

[0033] After PCR, 5 μL of the amplification product was subjected to agarose gel electrophoresis to confirm the amplification of bands ranging from 250 to 520 bp. Then, capillary electrophoresis was performed using an ABI 3730XL to detect the genotyping of each locus. Figures 2-3The genotyping results show that each amplification site obtained a stable and accurate peak signal. The peaks are neat and clear, the main peak is prominent, the baseline is stable, and no obvious spurious peaks or non-specific interference peaks are observed. The interpretation is good. If the peak signal reaches the set threshold and the allele length conforms to the repetitive motif rule, the genotyping is stable and can be used for population detection.

[0034] (4) The peak shape was converted into alleles using the software GeneMarker V2.2.2.0. Table 7 shows the genetic parameters of the sample population.

[0035] Table 7. Genetic parameters of 10 microsatellite loci in the saddle-banded grouper. Note: Primer: locus, N: number of individuals, Na: number of alleles, Ne: effective number of alleles, I: Shannon information index, Ho: observed heterozygosity; He: expected heterozygosity; uHe: unbiased expected heterozygosity; F: fixation index.

[0036] The above results demonstrate that the SSR markers provided by this invention exhibit good polymorphism and can effectively amplify target loci in *Gymnocypris saddleback*. The multiplex PCR method using 10 primer pairs is stable and accurate in *Gymnocypris saddleback* population genotyping, improving detection efficiency and reducing detection costs, thus meeting the requirements for genetic diversity analysis, germplasm resource assessment, and fisheries resource development and protection in *Gymnocypris saddleback* populations.

[0037] The above description is only a non-limiting embodiment of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the inventive concept of the present invention and without creative effort, and these all fall within the protection scope of the present invention.

Claims

1. A set of SSR fluorescently labeled primers for multiplex PCR of saddleback grouper, characterized in that: The SSR fluorescently labeled primer set for multiplex PCR of saddle-banded grouper consists of specific primer pairs and universal primer pairs; wherein, the specific primer pairs are G1 group and / or G2 group, G1 group is primer pair E1-01-E1-05 shown in SEQ ID NO: 1-10, and G2 group is primer pair E1-06-E1-10 shown in SEQ ID NO: 11-20.

2. The SSR fluorescently labeled primer set for multiplex PCR of saddle-banded grouper according to claim 1, characterized in that: Each primer pair consists of a forward primer F and a reverse primer R; where: The base sequences of the forward and reverse primers for primer pair El-01 are shown in SEQ ID NO: 1-2, respectively; The base sequences of the forward and reverse primers for primer pair El-02 are shown in SEQ ID NO: 3-4, respectively; The base sequences of the forward and reverse primers for primer pair E1-03 are shown in SEQ ID NO: 5-6, respectively; The base sequences of the forward and reverse primers for primer pair E1-04 are shown in SEQ ID NO: 7-8, respectively; The base sequences of the forward and reverse primers for primer pair E1-05 are shown in SEQ ID NO: 9-10, respectively; The base sequences of the forward and reverse primers for primer pair E1-06 are shown in SEQ ID NO: 11-12, respectively; The base sequences of the forward and reverse primers for primer pair E1-07 are shown in SEQ ID NO: 13-14, respectively; The base sequences of the forward and reverse primers for primer pair E1-08 are shown in SEQ ID NO: 15-16, respectively; The base sequences of the forward and reverse primers for primer pair El-09 are shown in SEQ ID NO: 17-18, respectively; The base sequences of the forward and reverse primers for primer pair E1-10 are shown in SEQ ID NO: 19-20, respectively.

3. The SSR fluorescently labeled primer set for multiplex PCR of saddle-banded grouper according to claim 1, characterized in that: The universal primer pair is fluorescently labeled universal primers M13 and PQE-F; wherein the base sequence of M13 is shown in SEQ ID NO: 21, and the base sequence of PQE-F is shown in SEQ ID NO:

22.

4. The application of the SSR fluorescently labeled primer set for multiplex PCR of saddle-banded grouper as described in claim 1, characterized in that: Application of the SSR fluorescently labeled primer set for multiplex PCR of saddle-banded grouper in germplasm resource evaluation and pedigree identification of saddle-banded grouper; Alternatively, the SSR fluorescently labeled primer set for multiplex PCR of the saddle-banded grouper may be used to assess the genetic diversity of the saddle-banded grouper and to promote the development and protection of fishery resources.

5. A kit for multiplex PCR of saddleback grouper, characterized in that: The kit contains the SSR fluorescently labeled primer set for multiplex PCR of saddle-banded grouper as described in claim 1.

6. The application of the reagent kit according to claim 5, characterized in that: Application of the kit in germplasm resource evaluation and pedigree identification of saddle-banded grouper; Alternatively, the kit may be used to assess the genetic diversity of saddleback grouper and to promote the development and conservation of fishery resources.

7. An SSR typing method for genetic diversity in saddleback grouper, characterized in that: (1) Extract genomic DNA from the sample to be tested; (2) The genomic DNA in step (1) is amplified by multiplex PCR using the specific primer pair in claim 1 to obtain the amplification product; (3) The amplification products were subjected to agarose gel electrophoresis to amplify specific bands with a length range of 250-520bp and a gradient. The sample to be tested contained saddle-banded grouper. At the same time, the genotype was performed based on the products. The genotype information was used for the genetic diversity analysis of the saddle-banded grouper population.

8. The SSR typing method for genetic diversity analysis of saddleback grouper according to claim 7, characterized in that: In step (2), the specific primer pair of claim 1, namely the primer pair E1-01 to E1-05 (G1 group), is used. The total volume of the G1 group multiplex PCR amplification reaction system is 25 μL, including: 1.4-1.6 μL of 5 specific primer pairs; 0.68-0.76 μL of universal primers; 100 ng of template DNA; 12.5 μL of Taq HS (Takara); and 8.1-8.4 μL of other reaction components, which are then added to a total of 25 μL.

9. The SSR typing method for genetic diversity analysis of saddleback grouper according to claim 7, characterized in that: In step (2), the specific primer pair of claim 1, namely the primer pair E1-06-E1-10 (G2 group), is used. The total volume of the G2 group multiplex PCR amplification reaction system is 25 μL, including: 1.4-1.6 μL of 5 specific primer pairs; 0.68-0.76 μL of universal primers; 100 ng of template DNA; 12.5 μL of Taq HS (Takara); and 8.1-8.4 μL of other reaction components, which are then added to a total of 25 μL.

10. The SSR typing method for genetic diversity analysis of saddleback grouper according to claim 8 or 9, characterized in that: In the multiplex PCR amplification reaction system, the volume ratio of the forward primer to the reverse primer in each primer pair is 1:4.

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