Star point SSR multiplex PCR primer and application thereof

Abstract

The application discloses a multiplex PCR primer for SSR of Sparus macrocephalus and application thereof, the multiplex PCR primer for SSR of Sparus macrocephalus comprises eight pairs of specific primers, namely primer pairs XD1, XD2, XD3, XD4, XD5, XD6, XD7 and XD8, and base sequences of the eight pairs of specific primers are shown in SEQ ID NO. 1-16 in sequence respectively; a multiplex fluorescence PCR method for SSR of Sparus macrocephalus and application of the multiplex PCR primer, a kit containing the primer or a method in evaluation of genetic diversity of Sparus macrocephalus are also disclosed; the primer amplification in the application is 3-6 base repeats of SSR sites, is stable and accurate in typing, and the method comprises a group of eight microsatellite sites, is high in accuracy, low in cost and convenient to operate.

Description

Technical Field

[0001] This invention belongs to the field of SSR marker technology, specifically relating to a multiplex PCR primer for SSR of the spotted snapper and its application. Background Technology

[0002] The spotted snapper (Lutjanus stellates), also known as the stone clam or yellow-finned snapper, belongs to the order Perciformes, family Lutjanidae, and genus Lutjanus. It is popular among consumers for its golden-yellow fins, delicious flesh, and rich nutritional value, and is a common target for coastal fishing. However, due to overfishing in recent years, the wild population of spotted snapper has declined, leading to widespread artificial breeding efforts.

[0003] Molecular markers are specific DNA fragments that reflect certain differences in the genomes of individuals or populations. They are characterized by high polymorphism, co-dominance, and good stability, playing a crucial role in genetic diversity analysis, kinship and species identification, genetic mapping, and gene localization. SSRs (microsatellites), also known as simple sequence repeats, are DNA sequences in the genome composed of multiple tandem repeats of 1-6 nucleotides as core units. SSR multiplex PCR technology is an important tool in many fields, including paternity testing, population genetics analysis, and breeding. Summary of the Invention

[0004] The first objective of this invention is to provide SSR multiplex PCR primers and kits for spotted snapper, which amplify SSR sites with 3-6 base repeats, resulting in stable and accurate genotyping.

[0005] The present invention also aims to provide a method for SSR multiplex fluorescent PCR of spotted snapper, which includes a set of 8 SSR loci. This method is highly accurate, low in cost and easy to operate.

[0006] The final object of the present invention is to provide the application of the above-described SSR multiplex PCR primers, the above-described kits, or the above-described methods in assessing the genetic diversity of spotted snapper.

[0007] The first objective of the present invention is achieved by the following technical solution: a multiplex PCR primer for spotted snapper SSR, the multiplex PCR primer comprising 8 pairs of specific primers, the 8 pairs of specific primers being primer pairs XD1, XD2, XD3, XD4, XD5, XD6, XD7 and XD8, wherein each primer pair includes a forward primer and a reverse primer, and the base sequences of the 8 pairs of specific primers are shown in sequence as SEQ ID NO.1~16.

[0008] Furthermore, it also includes four fluorescently labeled universal primers: M13, PQE-F, RV3, and pVP16.

[0009] In some preferred embodiments of the present invention, the 5' end of the forward primers of primer pairs XD1, XD2, and XD3 is connected to the universal primer M13; the 5' end of the forward primers of primer pairs XD4 and XD5 is connected to the universal primer PQE-F; the 5' end of the forward primers of primer pairs XD6 and XD7 is connected to the universal primer RV3; and the 5' end of the forward primer of primer pair XD8 is connected to the universal primer pVP16. However, this is not a limitation of the present invention, and the universal primers can be selected according to specific circumstances.

[0010] The base sequences of the universal primers M13, PQE-F, RV3 and pVP16 are shown in SEQ ID NO.17~20, respectively.

[0011] In some preferred embodiments of the present invention, the fluorescent label that cooperates with the universal primer M13 is FAM, the fluorescent label that cooperates with the universal primer PQE-F is HEX, the fluorescent label that cooperates with the universal primer RV3 is ROX, and the fluorescent label that cooperates with the universal primer pVP16 is TAMRA. However, this is not a limitation of the present invention. The fluorescent labels can be selected according to specific circumstances. In addition, theoretically, these fluorescent labels can also be interchanged.

[0012] The present invention also provides a kit for SSR multiplex PCR of spotted snapper, including the aforementioned primers for SSR multiplex PCR of spotted snapper.

[0013] Furthermore, the kit for SSR multiplex PCR of spotted snapper provided by the present invention includes, in addition to the aforementioned primers for SSR multiplex PCR of spotted snapper, four fluorescently labeled universal primers M13, PQE-F, RV3 and pVP16.

[0014] The second objective of this invention is achieved through the following technical solution: a method for SSR multiplex fluorescent PCR of spotted snapper, comprising the following steps:

[0015] (1) Extraction of DNA from spotted snapper: Genomic DNA was extracted from spotted snapper;

[0016] (2) Multiplex fluorescent PCR amplification: The genomic DNA in step (1) is amplified by multiplex fluorescent PCR using the SSR multiplex PCR primers or the kit described above to obtain the amplification product;

[0017] (3) Genetic diversity assessment: The amplification products were genotyped using capillary electrophoresis, and the alleles of each locus and individual were counted. Genetic diversity was compared based on the heterozygosity index.

[0018] In the above method of multiplex fluorescent PCR for SSR of spotted snapper:

[0019] Preferably, the reaction system for multiplex fluorescent PCR amplification in step (2) is as follows: 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, 1.2 μL of 10 μM fluorescently labeled universal primer M13, 0.8 μL of 10 μM fluorescently labeled universal primer PQE-F, 0.8 μL of 10 μM fluorescently labeled universal primer RV3, 0.4 μL of 10 μM fluorescently labeled universal primer pVP16, 1.5 μL bovine serum albumin (BSA), and 14.3 μL ultrapure water, for a total system volume of 50 μL.

[0020] Preferably, the reaction program for multiplex fluorescent PCR amplification in step (2) is as follows: pre-denaturation at 95℃ for 5 min; 27 cycles of 95℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s; 8 cycles of 95℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s; and finally extension at 72℃ for 30 min, followed by storage at 4℃.

[0021] Preferably, in step (3), a gene analyzer (e.g., ABI3730XL) is used to perform genotyping on the amplification products and read the individual genotype.

[0022] The method of SSR multiplex fluorescent PCR for spotted snapper of the present invention first screens a set of SSR amplification primer combinations, which contains a total of 8 pairs of specific primers; then, each pair of specific primers and fluorescently labeled universal primers are added to a reaction tube, the target fragment is amplified by PCR, and the amplification products of different primers are separated by capillary electrophoresis. Finally, the separated bands are statistically analyzed.

[0023] The last objective of the present invention is achieved by the following technical solution: the application of the above-described SSR multiplex PCR primers, the above-described kits, or the above-described methods in assessing the genetic diversity of spotted snapper.

[0024] The above-mentioned method for SSR multiplex fluorescent PCR of spotted snapper of the present invention uses a reliable and effective combination of SSR primers and employs multiplex PCR and capillary electrophoresis techniques to genotype spotted snapper populations. In addition to being used to assess the genetic diversity of spotted snapper, it can also be further used for parentage identification, population genetics research, germplasm resource identification, pedigree identification, etc.

[0025] The present invention has the following advantages:

[0026] (1) The SSR multiplex PCR primers for spotted snapper provided by the present invention have high polymorphism of amplification sites;

[0027] (2) The SSR multiplex PCR primers for spotted snapper provided by the present invention amplify SSR sites with 3-6 base repeats, and the typing is stable and accurate;

[0028] (3) The method for multiplex fluorescent PCR of starfish SSRs provided by the present invention includes a set of 8 SSR loci. The method has high accuracy and low cost.

[0029] (4) In addition to assessing the genetic diversity of spotted snapper, the primers, kits or methods of this invention can also be used for parentage identification, genetic research, germplasm resource identification and population pedigree identification of spotted snapper populations, and can be further promoted and applied in the evaluation of the effects of stock enhancement and release. Attached Figure Description

[0030] Figure 1 This is an agarose gel electrophoresis image of the PCR amplification products of the eight microsatellite loci of the spotted snapper in Example 2. The loci from left to right in the image are: Marker, XD1, XD2, XD3, XD4, XD5, XD6, XD7 and XD8. Figure 2 The images show the genotype diagrams for the Lst5, Lst14, and Lst15 samples genotyped using the XD1, XD2, and XD3 microsatellite loci of *Syngonium spp.* in Example 2. Image a shows the genotype diagram for the Lst5 sample genotyped using the XD1, XD2, and XD3 microsatellite loci of *Syngonium spp.* Image b shows the genotype diagram for the Lst14 sample genotyped using the XD1, XD2, and XD3 microsatellite loci of *Syngonium spp.* Image c shows the genotype diagram for the Lst15 sample genotyped using the XD1, XD2, and XD3 microsatellite loci of *Syngonium spp.* Figure 3 The images show the genotype diagrams for the Lst5, Lst14, and Lst15 samples genotyped using the XD4 and XD5 microsatellite loci of *Syngonium spp.* in Example 2. Image a shows the genotype diagram for the Lst5 sample genotyped using the XD4 and XD5 microsatellite loci of *Syngonium spp.*, image b shows the genotype diagram for the Lst14 sample genotyped using the XD4 and XD5 microsatellite loci of *Syngonium spp.*, and image c shows the genotype diagram for the Lst15 sample genotyped using the XD4 and XD5 microsatellite loci of *Syngonium spp.* Figure 4The images show the genotype diagrams for the Lst5, Lst14, and Lst15 samples genotyped using the XD6 and XD7 microsatellite loci of *Stellaria media* in Example 2. Specifically, a) is the genotype diagram for the Lst5 sample genotyped using the XD6 and XD7 microsatellite loci of *Stellaria media*, b) is the genotype diagram for the Lst14 sample genotyped using the XD6 and XD7 microsatellite loci of *Stellaria media*, and c) is the genotype diagram for the Lst15 sample genotyped using the XD6 and XD7 microsatellite loci of *Stellaria media*. Figure 5 The images show the genotype diagrams for the Lst5, Lst14, and Lst15 samples genotyped using the XD8 microsatellite locus of *Syngonium spp.* in Example 2. Image a shows the genotype diagram for the Lst5 sample genotyped using the XD8 microsatellite locus of *Syngonium spp.*, image b shows the genotype diagram for the Lst14 sample genotyped using the XD8 microsatellite locus of *Syngonium spp.*, and image c shows the genotype diagram for the Lst15 sample genotyped using the XD8 microsatellite locus of *Syngonium spp.* Detailed Implementation

[0031] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. However, it should be noted that the following embodiments are only used to describe the content of the invention and do not constitute a limitation on the scope of protection of the present invention. Unless otherwise specified, the reagents or materials used in the embodiments are all from commercial sources. Unless otherwise specified, the experimental instruments used are all conventional laboratory instruments.

[0032] Example 1

[0033] The SSR multiplex PCR primers for spotted snapper provided in this embodiment include 8 pairs of specific primers, namely primer pairs XD1, XD2, XD3, XD4, XD5, XD6, XD7 and XD8. Each primer pair includes one forward primer and one reverse primer. The base sequences of the 8 pairs of specific primers are shown in SEQ ID NO.1~16 respectively.

[0034] It also includes four universal fluorescently labeled primers: FAM for M13, HEX for PQE-F, ROX for RV3, and TAMRA for pVP16. The base sequences of the universal primers M13, PQE-F, RV3, and pVP16 are shown in SEQ ID NO.17~20, respectively.

[0035] The 5' end of the forward primers of primer pairs XD1, XD2, and XD3 is connected to the universal primer M13; the 5' end of the forward primers of primer pairs XD4 and XD5 is connected to the universal primer PQE-F; the 5' end of the forward primers of primer pairs XD6 and XD7 is connected to the universal primer RV3; and the 5' end of the forward primer of primer pair XD8 is connected to the universal primer pVP16.

[0036] The primer design process is as follows:

[0037] HiFi sequencing and Hi-C sequencing technologies were used to obtain the whole genome sequence and the sequence of long-range interaction sites, respectively. After assembly, the chromosome-level genome reference sequence of the starfish snapper was obtained. Based on the genome reference sequence, SSRs on the genome were genotyped, and SSR sites with high polymorphism and motif lengths of 3-6 bases were screened. Primers were designed using Primer3, and after evaluating the amplification specificity of the primers and the compatibility between primer combinations, a set of multiplex SSR-PCR combinations was selected, containing the sequences of 8 SSR sites (the sequences of the fragments containing sites XD1, XD2, XD3, XD4, XD5, XD6, XD7, and XD8) and 8 pairs of specific primers (sequences shown in SEQ ID NO. 1~16). The 8 pairs of specific primers are shown in Table 1 below.

[0038] Table 1. Specific primer pairs and fluorescently labeled universal primers for SSR site amplification

[0039] The sequence of the segment containing site XD1: TACCTGCTGGTGTCATTACTGGTGCGGGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAACAGGCATCGCTGCAGTGTGTTTGACTCTAATCCAGGCTGACA (SEQ ID NO. 21); The sequence of the segment containing site XD2: TTCAAGCATAAGCAGTTACGGACAATGAATGAATGAATGAATGAATGAATGAATGAATGAATGAATGAATGAATGGATGGATGTAACTCCCAATGATGGTGATTTTATAGTCATGTTGCGGGCTGTTTGTTAAAAATTCTGCAGTTCTTTCCGACAAGCAGGCAATATTGTCACTGTCAGAAATTCCTGACATCTCTCATAATTAAAGACTTAATTATGAGATTAATTAATGATTAAAAATAATTAAAAATATAGGCTGACTTGAGCGTTTTTATTATGACTGGGCTGTTCATAATCAT (SEQ ID NO.22); Sequence of the fragment where locus XD3 is located: ACACTGCTCCTGCACTCTACTGGAGGGTCTGAGGAGGGGCTTTTAAACTCAGCTGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAAATATTACCACTTAAGAATACGGAAATCTAATCTGGTATTTACATCACCACAGTATGCATATGTAGGCTTATGTTTTTATTTGTGGGGTTAATTTTGTTCATATAGATGTCTAGACACTTGGATGCCATATTTTCATCTTGTACTTGGAGTAGATTATAAAATATTAAGGTAGAAAATTCAAGATATTTTTTAGTACCCGCCTTTGATGTGATATGACATATGCCAGTGCTTACATTTCTAATATATATCTATCAGTCCTCTGTCTTTACACACATGCATACAGCTGTTTGTCTGATTAACATTGCCTGG (SEQ ID NO.23); Sequence of the fragment where locus XD4 is located: TTGATATTGCAATTGTTCTGCCATAACAGTGTACTGTAAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATCAACTTTAAAGTTAGTGTTTGGACTACCTTAAAATTTTAGGTTGACTGCATTTGCAAC (SEQ ID NO.24); Sequence of the fragment where locus XD5 is located: CAATGGCTTGGTATCCCATACTCCGATAGAAGTCCCCACAAGGGTCCAAGATGATAAGAGCCGACTCTCTGGCCCATCCATCCATCCATCCATCCATCCATCCATCCATCCATTTTCAACTGCTTGTCCGAAATCGGGTCGCGGAGGTAACAGTTCCAGCAGGGGACCCCAGACTTCCCTTTCCCCGACTACATTAGCCAGCTCTGACTGGGGGATCCTGAGGCATTCCCAGGCCAAGGCAGAGATGTAATCCCTGCACCTAGTCCT (SEQ ID NO.25); Sequence of the fragment where locus XD6 is located: CGTGGAAAGAAATATCCTCAGCATCAAAAGAAGTGACAATGACAGACTGTACCGACTGTGGAAAAGAATTTCTTCCCTGAGGACAAATTATCTATCTATCTAGCTATCTAGCTATCTAGCTATCTAGCTATCTAGCTTCCTTCATTCCTCCCTTCCTTCCTTCCTTCCTTCCTTCCTTCCTTCCTTCTTTGGGTATGAGCTGAAATGGAAAG (SEQ ID NO.26); Sequence of the fragment where locus XD7 is located: TATCTGCCCTACACCACTCCTTTTGGCATGGTCATTCCATGTGAAGCTCTGCCTTGTACGCTGTCCTGTGACATTTTACATGTGTTTTCATTGGTCTACATACCCTGTGATGTGGACAAAGTACTATGGCCAAATCCAGCAA GGCGAAGAGATGATGATGATGATGATGATGATGATGATGATTTTGTGATTCAAGCTTTCTTTTTGTTTCTACAAATGTTTCAGCACATGTGTGTAATCTTTGGAAATGTATGGATGTATTGTGTGCTGTGTTTGTCCA (SEQ ID NO.27); The sequence of the segment containing site XD8: GATTTGGTTGGTGTAGTCCAAATATTCTGCAAAAGTGTTGCAACCGTCCCCAGTCTCCCCTACTACTCTGTGTTGCTCATATCAGAAACATTGAAAGAACTTTACTTTGAACTCGATCCAAACATCAGTCAGTGAGTGAGTGAGTGAGTGAGTGAGTGAGTGAGTGAGTGAGTGAGTGAGTGAGTGAGTGAGTGAGTGAGTGAGTGGTGTTCCAGTGTTCAGTCTTACCCGTCTCGAT (SEQ ID NO.28).

[0040] Alternatively, a kit for SSR multiplex PCR of starfish can be prepared by adding 8 pairs of specific primers or 8 pairs of specific primers with four fluorescently labeled universal primers M13, PQE-F, RV3 and pVP16, along with conventional reagents in the field.

[0041] Example 2

[0042] The method for multiplex fluorescent PCR of spotted snapper SSR provided in this embodiment includes the following steps:

[0043] (1) Extraction of DNA from spotted snapper: 29 caudal fins (Lst1~Lst29) of spotted snapper were cut and genomic DNA was extracted;

[0044] Genomic 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.

[0045] (2) Multiplex fluorescent PCR amplification: The genomic DNA in step (1) was amplified by multiplex fluorescent PCR using the starfish SSR multiplex PCR primers or kit in Example 1 to obtain the amplification product;

[0046] The multiplex fluorescent PCR amplification reaction system in step (2) is as follows: 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, 1.2 μL of 10 μM fluorescently labeled universal primer M13, 0.8 μL of 10 μM fluorescently labeled universal primer PQE-F, 0.8 μL of 10 μM fluorescently labeled universal primer RV3, 0.4 μL of 10 μM fluorescently labeled universal primer pVP16, 1.5 μL bovine serum albumin (BSA), 14.3 μL ultrapure water, and a total system volume of 50 μL.

[0047] The multiplex fluorescent PCR amplification reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s, 27 cycles; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s, 8 cycles; and finally, extension at 72℃ for 30 min, followed by storage at 4℃.

[0048] (3) The multiplex fluorescent PCR products were genotyped on an automated sequencer (ABI 3730XL) and the individual genotypes were read; After PCR, 5 µL was electrophoresed on an agarose gel to detect diffuse bands of the expected size, and the rest were sent to a commercial company for genotyping using an ABI 3730XL. The electrophoresis diagram of the amplified bands at each site was verified using agarose gel electrophoresis, as shown below. Figure 1 As shown, from Figure 1 The images show that the bands amplified by the 8 pairs of primers are clear and bright, indicating the specificity of the primers.

[0049] Genotyping diagrams of three samples (Lst5, Lst14, and Lst15) using the microsatellite loci XD1, XD2, XD3, XD4, XD5, XD6, XD7, and XD8 of the star-spotted snapper are shown below. Figure 2-5 As shown, from Figures 2-5 The results show that the peaks at these eight loci are stable, allowing for batch testing of the population. In addition to assessing the genetic diversity of spotted snapper, it can also be used for parentage testing and pedigree construction in breeding.

[0050] The genotypes at each locus were read using GeneMarker V2.2.2.0 software. Table 2 shows the genotype data of the spotted snapper, and Table 3 shows the population genetic parameters of the spotted snapper.

[0051] Table 2 Alleles of Spotted Snapper

[0052]

[0053] Note: "0" in the table indicates that the reading failed.

[0054] Table 3. Genetic parameters of 8 SSR loci in the spotted snapper population.

[0055]

[0056] Note: Locus: locus, N: number of valid individuals, Na: number of alleles, Ne: number of valid alleles, I: Shannon information index, Ho: observed heterozygosity; He: expected heterozygosity; F: fixation index.

[0057] Tables 2 and 3 show that 8 sites have high polymorphism.

[0058] The above results demonstrate that the SSR multiplex fluorescent PCR method of this invention is stable and accurate in typing the spotted snapper population, meeting the requirements for parentage identification, germplasm identification, family management, and evaluation of the propagation and release effects of spotted snapper.

[0059] 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 multiplex PCR primer for SSR of spotted snapper, characterized in that, The SSR multiplex PCR primers include 8 pairs of specific primers, namely primer pairs XD1, XD2, XD3, XD4, XD5, XD6, XD7 and XD8, wherein each primer pair includes one forward primer and one reverse primer, and the base sequences of the 8 pairs of specific primers are shown in SEQ ID NO.1~16 respectively.

2. The star-spotted snapper SSR multiplex PCR primers according to claim 1, characterized in that, It also includes four fluorescently labeled universal primers M13, PQE-F, RV3 and pVP16, the base sequences of which are shown in SEQ ID NO.17~20 respectively.

3. A kit for SSR multiplex PCR of spotted snapper, characterized in that, Includes the SSR multiplex PCR primers for spotted snapper as described in claim 1.

4. The reagent kit according to claim 3, characterized in that, It also includes four fluorescently labeled universal primers M13, PQE-F, RV3 and pVP16, the base sequences of which are shown in SEQ ID NO.17~20 respectively.

5. A method for multiplex fluorescent PCR of SSRs of spotted snapper, characterized in that, Includes the following steps: (1) Extraction of DNA from spotted snapper: Genomic DNA was extracted from spotted snapper; (2) Multiplex fluorescent PCR amplification: The genomic DNA in step (1) is amplified by multiplex fluorescent PCR using the SSR multiplex PCR primers described in claim 2 or the kit described in claim 4 to obtain the amplification product; (3) Genetic diversity assessment: The amplification products were genotyped using capillary electrophoresis, and the alleles of each locus and individual were counted. Genetic diversity was compared based on the heterozygosity index.

6. The method for SSR multiplex fluorescent PCR of spotted snapper according to claim 5, characterized in that, The reaction system for multiplex fluorescent PCR amplification in step (2) is as follows: 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, 1.2 μL of 10 μM fluorescently labeled universal primer M13, 0.8 μL of 10 μM fluorescently labeled universal primer PQE-F, 0.8 μL of 10 μM fluorescently labeled universal primer RV3, 0.4 μL of 10 μM fluorescently labeled universal primer pVP16, 1.5 μL bovine serum albumin (BSA), and 14.3 μL ultrapure water, for a total system volume of 50 μL.

7. The method for SSR multiplex fluorescent PCR of spotted snapper according to claim 5, characterized in that, The reaction program for multiplex fluorescent PCR amplification in step (2) is as follows: pre-denaturation at 95℃ for 5 min; 27 cycles of 95℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s; 8 cycles of 95℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s; and finally, fluorescence extension at 72℃ for 30 min, followed by storage at 4℃.

8. The method for multiplex fluorescent PCR of spotted snapper SSR according to claim 5, characterized in that, In step (3), a gene analyzer is used to perform genotyping on the amplification products and read the individual genotype.

9. The use of the SSR multiplex PCR primers of claim 1 or 2, the kit of claim 3 or 4, or the method of any one of claims 5-8 in assessing the genetic diversity of spotted snapper.

Images