A method for identifying fish (fish mang) based on microsatellite markers, primers for identification, and applications thereof

Abstract

The present invention discloses a method for identifying fish (fish mang) based on microsatellite markers, as well as primers for identification and applications thereof. The identification primers include primer pairs Ps-Chr-4-1 and Py-Chr-4-2. The method extracts genomic DNA from individual samples of Su's round belly (fish mang), long silk (fish mang), and their hybrids, performs PCR amplification on the obtained DNA genome using the identification primers, and electrophoreses and develops the PCR amplification products using 1.5% agarose gel; and distinguishes Su's round belly (fish mang), long silk (fish mang), and hybrids based on the presence or absence of specific fragments. The method of the present invention is simple, efficient, rapid, accurate, and low-cost. Using this method, Su's round belly (fish mang), long silk (fish mang), and their hybrids can be quickly and accurately identified and distinguished. This method lays a technical foundation for the efficient batch identification of Su's round belly (fish mang), long silk (fish mang), and their hybrids, and can be promoted and applied in the germplasm identification and family genetic management of Su's round belly (fish mang), long silk (fish mang), and hybrids.

Description

Technical Field

[0001] The present invention belongs to the technical field of microsatellite markers, and in particular relates to a method for identifying fish (Stenoptera: fulvidraco) based on microsatellite markers, primers for identification thereof, and applications thereof. Background Art

[0002] Accurate identification of fish is crucial for ecological research, aquaculture, and biodiversity conservation. Fish, as the most primitive vertebrates, represent a large group of species that have hybridized throughout evolutionary history, demonstrating primitiveness, complexity, and diversity. Mitochondrial primers have limitations in identifying hybrid species, and the development of specific molecular markers is currently a key method for identifying fish species and for their conservation and development.

[0003] Su's round belly (fish mango) Pangasius sutchi or Pangasianodonhypophthalmus ) belongs to the order Siluriformes, family Gastropodidae, genus Adonta, also known as freshwater shark. This fish is a tropical omnivorous fish with advantages such as fast growth rate, strong disease resistance, tolerance to low oxygen, high meat yield, tender meat and no intramuscular spines. At the same time, the fish skin can be used to make gelatin, and the fish bones can be used to extract the medicinal ingredient chondroitin sulfate. It is an excellent freshwater aquaculture species. Pangasius sanitwongsei Smith The genus (Shrimp) (1931) belongs to the order Siluriformes, family Megalospermidae, and is native to the Chao Phraya and Mekong River basins. It prefers deep waters and is a carnivorous fish in its natural habitat, primarily feeding on fish, shrimp, crabs, and other aquatic animals. Spawning occurs between April and May. The long-striped mullet has tender, delicious meat and a fast growth rate, making it a highly economically valuable aquatic species. Furthermore, the long-striped mullet is highly valued for its ornamental value. Its broad head, agile swimming posture, and distinctive fins on its pectoral, pelvic, and dorsal fins make it a favorite among ornamental fish breeders.

[0004] Fish are the most numerous and diverse vertebrates alive today. It is currently believed that their origins involved multiple genome polyploidization and complex hybridization events, leading to the fusion of different genomes and the doubling of chromosome sets, resulting in rich genetic diversity. Distant hybridization in fish can promote polyploidization, and hybridization and polyploidization are of great significance to the genetic breeding and evolutionary research of fish. The offspring of fish hybridization—hybrid fish—sometimes inherit some genetic characteristics of both parents, even unfavorable traits, or sometimes acquire selected or favorable traits from one parent, or even possess superior traits that surpass both parents, such as rapid growth, good meat quality, strong disease resistance, and high environmental tolerance. This is known as hybrid vigor. To improve the germplasm resources of the Su's round belly (fish awn) and long silk (fish awn), crossbreeding has been carried out. While hybrid vigor is present, the hybrid offspring also show a high morphological similarity to both parents, which can lead to inaccurate morphological identification. Commonly used mitochondrial markers for species identification are limited in their inability to distinguish between the maternal and female species.

[0005] Identifying bioeconomically important sharks during port inspections is difficult (if not impossible) using traditional classification tools because heads and fins are removed during processing at sea. During processing, morphological and phylogenetic criteria crucial for accurate specimen identification are lost. Several different genetic identification methods have been developed to address misidentification or reveal endangered shark species caught. These include gel-based identification methods, DNA barcoding, and sequencing-based identification methods (using cytochrome b sequences and high-resolution melting analysis). Furthermore, recent studies have demonstrated the importance of cross-species microsatellites in identifying species-specific allele sizes and unique allele frequencies at multiple loci. Therefore, to better identify species of S. suis (fish tang), S. longissimus (fish tang), and their hybrids, there is an urgent need to develop molecular markers suitable for distinguishing hybrids from their parents, leveraging the stability, high reproducibility, and ease of use of microsatellite markers.

[0006] The PacBio Sequel II, a third-generation sequencing platform, utilizes SMRT sequencing technology for single-molecule real-time sequencing. SMRT sequencing utilizes SMRT cells, each of which is packed with millions of zero-mode waveguides (ZMWs). During sequencing, DNA polymerase and a template molecule are anchored at the bottom of a ZMW pore. Excitation light at the bottom of the pore excites fluorescent markers on the nucleotide substrate, which are then recorded by a monitoring system to reveal the base information. During the sequencing process, DNA molecules do not undergo PCR amplification, enabling individual sequencing of each DNA molecule. Its advantages include: A. long read length and high throughput; B. high consistency and accuracy; C. uniform coverage; D. high-precision, long-length HiFireads; and E. haploid assembly. Third-generation sequencing plays a crucial role in genome analysis and assembly, and its high resolution significantly improves the completeness of genome assemblies. The haplotypes of the hybrid parents were obtained using genomic haplotype assembly. Based on haplotype analysis, the specific sequences of the nuclear genome among the three were found, and molecular markers suitable for distinguishing hybrid fish from their parents were developed, which will help in efficient and accurate species identification in stocking, ports, customs, etc. Summary of the Invention

[0007] The present invention aims to provide a method for identifying fish (fish tang) based on microsatellite markers, as well as primers and applications for such identification. This method and primers can accurately identify and differentiate Su's round-bellied (fish tang), long-threaded (fish tang), and their hybrids, laying a technical foundation for efficient, large-scale identification of Su's round-bellied (fish tang), long-threaded (fish tang), and their hybrids.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The primers used for identifying fish (Yumang) based on microsatellite markers include two pairs of primers, Ps-Chr-4-1 and Py-Chr-4-2. The sequences of the primers are shown in Table 1 below:

[0010]

[0011] Furthermore, the sequences of the two pairs of primers Ps-Chr-4-1 and Py-Chr-4-2 are shown in SEQ ID NOs: 1 to 4, respectively.

[0012] The identification primers are used in identifying and distinguishing Su's round belly (fish mang), long silk (fish mang) and their hybrids.

[0013] On the other hand, the present invention also provides a method for identifying (fish mang) fish based on microsatellite markers, which is used to identify and distinguish Su's round belly (fish mang), long silk (fish mang) and their hybrids, specifically comprising the following steps:

[0014] (1) Extraction of genomic DNA from Su's round belly (fish mang), long silk (fish mang), and hybrids: Tail fin tissue samples were collected from Su's round belly (fish mang), long silk (fish mang), and their hybrids, and genomic DNA was extracted from each of them;

[0015] (2) Synthesize the two pairs of microsatellite marker primers mentioned above: Ps-Chr-4-1 and Py-Chr-4-2;

[0016] (3) PCR amplification: respectively taking the genomic DNA of Su's round belly (fish awn), long silk (fish awn), and hybrid obtained in step (1) and the two pairs of microsatellite marker primers synthesized in step (2) to perform PCR reaction;

[0017] (4) Genotyping: The PCR products were electrophoresed on a 1.5% agarose gel, using the DL2000 Maker as a DNA molecular weight standard reference, and the PCR product bands were developed and detected using a gel imaging system;

[0018] (5) (Fish awn) Determination of fish species: The fragment sizes of the PCR products of Ps-Chr-4-1 and Py-Chr-4-2 are 204 bp and 229 bp respectively; in the gel electrophoresis diagram of the test sample, if the PCR products of Ps-Chr-4-1 and Py-Chr-4-2 are both detected with bands, it is a hybrid individual; if the PCR product of Ps-Chr-4-1 has a band and the PCR product of Py-Chr-4-2 has no band, it is a long filament (fish awn); if the PCR product of Ps-Chr-4-1 has no band and the PCR product of Py-Chr-4-2 has a band, it is Su's round belly (fish awn).

[0019] Furthermore, in the above-mentioned method for identifying (fish tang) fish based on microsatellite markers, in step (1): collecting tail fin tissue samples of Su's round belly (fish tang), long silk (fish tang) and their hybrids, using high salt method, phenol / chloroform extraction method, commercial DNA extraction kit or magnetic bead method to obtain genomic DNA of each individual, and storing them for future use.

[0020] Further, in the method for identifying Pangasius hypophthalmus based on microsatellite markers, in step (3), the PCR reaction system is as follows: 6.25 μL of Taq enzyme PCR premix, 1.0 μL of template genomic DNA, 0.5 μL each of upstream and downstream amplification primers (concentration 10 μM), and add 4.25 μL of ddH2O to a total volume of 12.5 μL; the main components in the Taq enzyme PCR premix are 0.1 U / μl Taq DNA polymerase, 2X PCR reaction buffer, 3 mM MgCl2, and 0.4 mM dNTPs. The PCR reaction program is: pre-denaturation at 95°C for 10 min, denaturation at 95°C for 40 s, annealing at 55°C for 30 s, extension at 72°C for 60 s, for a total of 35 cycles, final extension at 72°C for 5 min, and preservation at 4°C.

[0021] Further, in the method for identifying Pangasius hypophthalmus based on microsatellite markers, in step (3), the concentrations of genomic DNA of Pangasianodon hypophthalmus, Pangasius sanitwongsei, and the hybrid are 50 ± 20 ng / μL, and the concentrations of upstream and downstream amplification primers are 10 μM.

[0022] Application of the method for identifying Pangasius hypophthalmus based on microsatellite markers in identifying and differentiating Pangasianodon hypophthalmus, Pangasius sanitwongsei, and their hybrids.

[0023] Note: The "Pangasius" in this article is a single Chinese character. Since this character is rare and cannot be displayed, it is represented by the two characters "Pangasius" in parentheses.

[0024] The beneficial effects of the present invention are as follows:

[0025] 1. The present invention discloses a pair of primers (Ps-Chr-4-1 and Py-Chr-4-2) for identifying Pangasius hypophthalmus based on microsatellite markers. Based on the parental genomic haplotypes in the hybrid as the foundation, genomic haplotype comparative analysis is carried out to search for heritable specific microsatellite markers of the parents. Primer design and verification are carried out based on this sequence fragment respectively. After population verification, it is found that these primers can accurately identify Pangasianodon hypophthalmus, Pangasius sanitwongsei, and their hybrids, with an accuracy rate of 100%.

[0026] 2. The present invention combines third-generation sequencing and second-generation sequencing to assemble high-quality parental haplotypes, obtains parental-specific microsatellite markers through parental haplotype comparison, and performs microsatellite genotyping based on agarose gel electrophoresis, achieving efficient and accurate species identification of Pangasianodon hypophthalmus, Pangasius sanitwongsei, and the hybrid. The present invention provides a new method for identifying Pangasius hypophthalmus, laying a technical foundation for batch identification of Pangasianodon hypophthalmus, Pangasius sanitwongsei, and their hybrid species.

[0027] 3. The two specific microsatellite markers screened by the present invention are mainly parent-specific and hereditary. Their amplification effect is stable, clear, and highly specific, providing a more objective basis for subsequent data regularization. Only two pairs of primers are used to complete the identification of (fish mang) fish, greatly reducing the cost and workload.

[0028] 4. The present invention discloses a method for identifying fish (fish mang) using microsatellite markers. This method involves extracting genomic DNA from individual samples of Su's round-bellied (fish mang), long-lined (fish mang), and hybrids. The obtained DNA is amplified by PCR using defined identification primers. The PCR amplification products are electrophoresed and visualized on a 1.5% agarose gel. The method then differentiates between Su's round-bellied (fish mang), long-lined (fish mang), and hybrids based on the presence or absence of specific fragments. The method is simple, efficient, rapid, and accurate, and can accurately identify and differentiate between Su's round-bellied (fish mang), long-lined (fish mang), and their hybrids. It can be widely applied in the identification of Su's round-bellied (fish mang), long-lined (fish mang), and hybrids, as well as in family genetic management.

[0029] 5. The present invention mainly uses stably expressed species-specific nuclear gene microsatellite markers as a species identification method, which can distinguish Su's round belly (fish awn), long silk (fish awn) and hybrids, overcoming the shortcomings of mitochondrial gene identification. Only two pairs of primers are used to complete the identification, thereby improving the working efficiency of microsatellite markers. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Gel electrophoresis detection results of the amplified products of Su's round belly (fish awn), long silk (fish awn) and their hybrids based on the Ps-Chr-4-1 primer; Figure 1 Among them, the PCR amplification products of primer Ps-Chr-4-1 showed bands in long silk (fish awn) and hybrids, but no bands in Su's round belly (fish awn);

[0031] Figure 2 Gel electrophoresis detection results of the amplified products of Su's round belly (fish awn), long silk (fish awn) and their hybrids based on the Py-Chr-4-2 primer; Figure 2 The PCR amplification products of Py-Chr-4-2 primers showed bands in Su's round belly (fish awn) and hybrids, but no bands in long filament (fish awn). DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to examples. Example 1

[0033] The identification of the heritable specific sequences of Su's round belly (fish awn) and long silk (fish awn) includes the following steps:

[0034] (1) Hybrid genome sequencing

[0035] Using the Sequel II third-generation sequencing platform, HiFireads, a sequencing method that combines long reads with high accuracy, was developed. The genome of a hybrid of Su's round belly (♀) x long silk (♂) was sequenced using CircμLar Consensus Sequencing (CCS) sequencing. Gaps in the third-generation sequencing results were filled using second-generation sequencing results. Genome assembly was performed using HiFiasm software to determine parental haplotypes. The genome assembly quality, accuracy, and completeness were assessed. The genome length was 808,852,175 bp, the N50 was 28,100,061 bp, the longest sequence length was 46,057,073 bp, and the GC content was 39.36%. The third-generation data were aligned to the assembled genome, with an alignment rate of 99.86% and a coverage of 99.99%. The second-generation data were aligned to the assembled genome, with an alignment rate of 99.83% and a coverage of 99.79%. 96.98% of the BUSCO genes were completely aligned, and high-quality haplotypes of both parental genomes were obtained.

[0036] (2) Parent-specific microsatellite search and primer design

[0037] In step (1), the parental haplotypes of the hybrid were obtained. Unique molecular markers were developed through haplotype comparative analysis. ① Collinearity analysis of the hap1 and hap2 genomes was performed using Mummer software. ② Collinearity variation detection was performed, with hap1 as the reference genome and hap2 as the qry genome. The structural variation types were identified using syri software and the results were statistically analyzed. ③ The SSRs unique to each parent were selected, and three sets of primers were designed using Primer 3 for each predicted SSR locus. 300 primer pairs with a fragment size of 200-300bp were screened, and 30 pairs were selected for primer synthesis and verification.

[0038] (3) Microsatellite marker population verification

[0039] Randomly select 5 each of Su's round belly (fish mang), long silk (fish mang) and hybrids as the validation population, use the primers obtained in step (2) for PCR amplification, and use DL2000 Marker to evaluate the size of the amplified fragment. Two pairs of primers that can 100% distinguish Su's round belly (fish mang), long silk (fish mang) and their hybrids were obtained. The primers are named Ps-Chr-4-1 and Py-Chr-4-2. The primer sequences are shown in Table 1. The details are as follows:

[0040] ① Extract genomic DNA from individual samples of Su's round belly (fish mang), long silk (fish mang), and hybrids: 5 samples each of Su's round belly (fish mang), long silk (fish mang), and hybrids were randomly selected, and genomic DNA was extracted using the high salt method. The specific steps are as follows: cut 0.5g of fin ray tissue, wash with double distilled water to remove the alcohol attached to the sample, cut it into pieces, and put it into a 1.5mL centrifuge tube; add 500μL of cell lysis buffer and 6μL of proteinase K to the centrifuge tube, digest it in a 55℃ water bath overnight, and shake it several times for the first 30 minutes; add 500μL of sodium chloride (4.5mol / L), 300μL of chloroform, mix it on a shaker at medium speed for 20 minutes, and centrifuge it at 13000rpm and 10℃ for 10 minutes; transfer the supernatant to a new tube (about 850μL), add 595μL of anhydrous isopropanol, mix it on a shaker at medium speed for 20 minutes, and centrifuge it at 13000rpm and 10℃ for 10 minutes, and discard the supernatant; add 500μL Digest the DNA with 75% ethanol in a 55°C water bath for 5 minutes, centrifuge at 13,000 rpm and 10°C for 20 minutes, and discard the supernatant. Dry the centrifuge tube in a clean bench for 1 hour, add 50-100 μL of TE solution (PH 8.0), dissolve the DNA at 4°C overnight, and store in a -20°C refrigerator for later use.

[0041] ② Screening for specific microsatellite markers: The 30 primer pairs obtained in the specific microsatellite search and primer design in step (2) above were screened and verified. PCR amplification was performed on 5 fish each of Su's round belly (fish mang), long silk (fish mang), and hybrids (genomic DNA extracted in step ①). Primers that could 100% distinguish Su's round belly (fish mang), long silk (fish mang), and their hybrids were screened. The PCR reaction system was: 6.25 μL 2 × Power Taq PCR premix (main components: 0.1 U / μL Taq DNA polymerase, 2X PCR reaction buffer, 3 mM MgCl2, and 0.4 mM dNTPs), 1.0 μL template genomic DNA (concentration 50±20 ng / μL), 0.5 μL each of upstream and downstream amplification primers (concentration 10 μM), and 4.25 μL ddH2O were added to a total volume of 12.5 μL. The PCR amplification procedure was as follows: 95°C pre-denaturation for 10 min, 95°C denaturation for 40 s, 55°C annealing for 30 s, 72°C extension for 60 s, for a total of 35 cycles, 72°C final extension for 5 min, and storage at 4°C. The PCR products were electrophoresed on a 1.5% agarose gel at 150 V for 30 min. Each gel was scanned and photographed using a gel imager. Two pairs of primers that could 100% distinguish Su's round belly (fish mang), long silk (fish mang) and their hybrids were obtained through screening and verification. The primers are named Ps-Chr-4-1 and Py-Chr-4-2, and the primer sequences are shown in Table 1. The gel electrophoresis images of the amplified products of PCR amplification of genomic DNA of Su's round belly (fish mang), long silk (fish mang) and their hybrids using primers Ps-Chr-4-1 and Py-Chr-4-2 are shown in Table 1. Figure 1 、 Figure 2 , Figure 1 The PCR amplification products of primer Ps-Chr-4-1 shown in the figure show bands in long silk (fish awn) and hybrids, but no bands in Su's round belly (fish awn); Figure 2 The PCR amplification products of Py-Chr-4-2 primers showed bands in Su's round belly (fish awn) and hybrids, but no bands in long filament (fish awn).

[0042]

[0043] Example 2

[0044] A method for identifying fish (fish mang) based on microsatellite markers, using this method to identify and distinguish Su's round belly (fish mang), long silk (fish mang) and their hybrids, specifically comprising the following steps:

[0045] (1) Extraction of genomic DNA from Su's round belly (fish fin), long silk (fish fin), and hybrids: Fin rays of Su's round belly (fish fin), long silk (fish fin), and their hybrids were collected, and genomic DNA of each individual was extracted using the high salt method. The specific extraction steps were the same as in Example 1, and the DNA was stored for future use.

[0046] (2) Two pairs of microsatellite marker primers were synthesized: Ps-Chr-4-1 and Py-Chr-4-2; the sequence information of the two pairs of primers is shown in Table 1.

[0047] (3) PCR amplification: the genomic DNA of Su's round belly (fish awn), long silk (fish awn), and hybrid obtained in step (1) and the two pairs of microsatellite marker primers synthesized in step (2) were respectively used for PCR reaction; the PCR reaction system was: 6.25 μL Taq enzyme PCR premix (main components are 0.1 U / μl Taq DNA polymerase, 2X PCR reaction buffer, 3mM MgCl2, and 0.4 mM dNTPs), 1.0 μL template genomic DNA (concentration 50±20ng / μL), 0.5 μL of upstream and downstream amplification primers (concentration 10uM), 4.25 μL ddH2O was added to a total volume of 12.5 μL; the PCR reaction program was: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 40s, 55℃ annealing for 30s, 72℃ extension for 60s, a total of 35 cycles, 72℃ final extension for 5 min, and storage at 4℃ to obtain PCR products.

[0048] (4) Genotyping: PCR products were electrophoresed on a 1.5% agarose gel, and each gel was scanned and photographed using a gel imager. PCR product bands were detected using a gel imaging system using a DL2000 Maker as a DNA molecular weight standard.

[0049] (5) (Fish awn) Determination of fish species: The fragment sizes of the PCR products of Ps-Chr-4-1 and Py-Chr-4-2 are 204 bp and 229 bp respectively; in the gel electrophoresis diagram of the test sample, if the PCR products of Ps-Chr-4-1 and Py-Chr-4-2 are both detected with bands, it is a hybrid individual; if the PCR product of Ps-Chr-4-1 has a band and the PCR product of Py-Chr-4-2 has no band, it is a long filament (fish awn); if the PCR product of Ps-Chr-4-1 has no band and the PCR product of Py-Chr-4-2 has a band, it is Su's round belly (fish awn).

[0050] Example 3

[0051] A method for identifying fish (fish mang) based on microsatellite markers, using this method to identify and distinguish Su's round belly (fish mang), long silk (fish mang) and their hybrids, specifically comprising the following steps:

[0052] (1) Extraction of genomic DNA from Su's round belly (fish mang), long silk (fish mang), and hybrids: Tissue samples of Su's round belly (fish mang), long silk (fish mang), and their hybrids were collected, and the genomic DNA of each individual was extracted using the phenol / chloroform extraction method, a commercial DNA extraction kit, or the magnetic bead method, and stored for later use.

[0053] (2) Two pairs of microsatellite marker primers were synthesized: Ps-Chr-4-1 and Py-Chr-4-2; the sequence information of the two pairs of primers is shown in Table 1.

[0054] (3) PCR amplification: the genomic DNA of Su's round belly (fish awn), long silk (fish awn), and hybrid obtained in step (1) and the two pairs of microsatellite marker primers synthesized in step (2) were respectively used for PCR reaction; the PCR reaction system was: 6.25 μL Taq enzyme PCR premix (main components are 0.1 U / μl Taq DNA polymerase, 2X PCR reaction buffer, 3mM MgCl2, and 0.4 mM dNTPs), 1.0 μL template genomic DNA (concentration 50±20ng / μL), 0.5 μL of upstream and downstream amplification primers (concentration 10uM), 4.25 μL ddH2O was added to a total volume of 12.5 μL; the PCR reaction program was: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 40s, 55℃ annealing for 30s, 72℃ extension for 60s, a total of 35 cycles, 72℃ final extension for 5 min, and storage at 4℃ to obtain PCR products.

[0055] (4) Genotyping: PCR products were electrophoresed on a 1.5% agarose gel, and each gel was scanned and photographed using a gel imager. PCR product bands were detected using a gel imaging system using a DL2000 Maker as a DNA molecular weight standard.

[0056] (5) (Fish awn) Determination of fish species: The fragment sizes of the PCR products of Ps-Chr-4-1 and Py-Chr-4-2 are 204 bp and 229 bp respectively; in the gel electrophoresis diagram of the test sample, if the PCR products of Ps-Chr-4-1 and Py-Chr-4-2 are both detected with bands, it is a hybrid individual; if the PCR product of Ps-Chr-4-1 has a band and the PCR product of Py-Chr-4-2 has no band, it is a long filament (fish awn); if the PCR product of Ps-Chr-4-1 has no band and the PCR product of Py-Chr-4-2 has a band, it is Su's round belly (fish awn).

Claims

1. A primer for identifying fish mackerel based on microsatellite markers, characterized in that: The primers include two pairs of primers, Ps-Chr-4-1 and Py-Chr-4-2, and the primer sequences are as follows: ； The fish mackerel is Su's round-bellied fish mackerel, long-thread fish mackerel and their hybrids.

2. The use of the identification primer as claimed in claim 1 in identifying and distinguishing Su's round-bellied fish awn, long-threaded fish awn and their hybrids.

3. A method for identifying fish mackerel based on microsatellite markers, characterized in that: The method is used to identify and differentiate Su's round-bellied fish mango, long-threaded fish mango and their hybrids, specifically comprising the following steps: (1) Extraction of genomic DNA from Su's round-bellied fish, long-line fish, and hybrids: Tail fin tissue samples were collected from Su's round-bellied fish, long-line fish, and their hybrids, and genomic DNA was extracted from each sample. (2) Synthesizing the two pairs of microsatellite marker primers described in claim 1: Ps-Chr-4-1 and Py-Chr-4-2; (3) PCR amplification: respectively taking the genomic DNA of the Su's round-bellied fish awn, the long-threaded fish awn, and the hybrid obtained in step (1) and the two pairs of microsatellite marker primers synthesized in step (2) to perform PCR reaction; (4) Genotyping: The PCR products were electrophoresed on a 1.5% agarose gel, using the DL2000 Maker as a DNA molecular weight standard reference, and the PCR product bands were developed and detected using a gel imaging system; (5) Identification of the species of mullet: The fragment sizes of the PCR products of Ps-Chr-4-1 and Py-Chr-4-2 are 204 bp and 229 bp, respectively. In the gel electrophoresis diagram of the test sample, if the PCR products of Ps-Chr-4-1 and Py-Chr-4-2 both have bands, it is a hybrid individual. If the PCR product of Ps-Chr-4-1 has a band and Py-Chr-4-2 has no band, it is a long-threaded mullet. If the PCR product of Ps-Chr-4-1 has no band and Py-Chr-4-2 has a band, it is a Su's round-bellied mullet.

4. The method for identifying fish sturgeon based on microsatellite markers according to claim 3, wherein In step (1): caudal fin tissue samples of Su's round-bellied fish, long-threaded fish and their hybrids are collected, and genomic DNA of each individual is extracted using a high salt method, a phenol / chloroform extraction method, a commercial DNA extraction kit or a magnetic bead method, and stored for later use.

5. The method for identifying fish sturgeon based on microsatellite markers according to claim 3, wherein In step (3), the PCR reaction system is: 6.25 μL Taq enzyme PCR premix, 1.0 μL template genomic DNA, 0.5 μL each of upstream and downstream amplification primers, concentration 10uM, and 4.25 μL ddH2O to a total volume of 12.5 μL.

6. The method for identifying fish sturgeon based on microsatellite markers according to claim 3, wherein In step (3), the PCR reaction procedure is: pre-denaturation at 95°C for 10 min, denaturation at 95°C for 40 s, annealing at 55°C for 30 s, extension at 72°C for 60 s, for a total of 35 cycles, final extension at 72°C for 5 min, and storage at 4°C.

7. The method for identifying fish sturgeon based on microsatellite markers according to claim 3, wherein In step (3), the concentration of genomic DNA of Su's round-bellied fish awn, long-threaded fish awn, and hybrid was 50±20 ng / μL, and the concentration of upstream and downstream amplification primers was 10 uM.

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