Method for rapidly identifying genetic composition of filial generation of megalobrama amblycephala and culter alburnus, specific primer pool and application

Abstract

The invention belongs to the field of molecular biology and animal molecular breeding, and discloses a method for identifying genetic composition of a hybrid generation of megalobrama amblycephala and culter alburnus, a specific primer pool and application of the specific primer pool in identifying the genetic composition of the hybrid generation of megalobrama amblycephala and culter alburnus. According to the invention, qualitative and quantitative detection is carried out on the basic group condition of SNP sites on each pair of homologous chromosomes of megalobrama amblycephala and culter alburnus by innovatively utilizing a method of combining multiple PCR and high-throughput sequencing, so that an efficient identification technology is provided for genetic composition of filial generations obtained from other related species such as megalobrama amblycephala and culter alburnus. Compared with a whole genome sequencing gene, the method has the advantages that only a specific region on each chromosome is amplified and sequenced, so that the sequencing cost is effectively reduced, the analysis period is also shortened, and the method is an efficient technology for quickly identifying the genetic composition of a filial generation obtained by hybridizing sibling species such as megalobrama amblycephala and culter alburnus.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology and animal molecular breeding, and particularly relates to a method for identifying the genetic composition of hybrid offspring of Megalobrama amblycephala and Culter alba, a specific primer pool and application. Background Art

[0002] The Chinese bream is endemic to medium-sized lakes in the middle and lower reaches of the Yangtze River in China. Since the success of artificial breeding technology in 1964, the Chinese bream has become the main force in China's freshwater aquaculture. This fish is favored by farmers for its wide diet, low breeding costs, fast growth rate, high survival rate, easy fishing and easy breeding. The annual production of the Chinese bream is stable at between 700,000 and 800,000 tons. It is a herbivorous fish known for its high back and short body. The Culter albus is widely distributed in the main streams, tributaries and affiliated lakes of major water systems in China. Since the breakthrough of artificial breeding technology in the mid-1990s, the Culter albus has become an indispensable part of freshwater aquaculture. This fish mainly feeds on plankton and is a carnivorous fish.

[0003] By using Megalobrama amblycephala and Culter alba as parents for reciprocal crosses, new breed lines with bisexual fertility can be successfully constructed. By backcrossing the F1 generation of these new breed lines with the original parents, fish varieties with significant hybrid advantages can be cultivated. For example, hybrid Culter alba (registration number of new aquatic species certificate: GS-02-003-2014), Hefang crucian carp, Xiangjun bream, etc., these varieties not only inherit excellent traits such as herbivory, rapid growth, tolerance to hypoxia and tender meat, but also have huge market potential.

[0004] Currently, the technologies that can identify the genotypes of hybrid offspring obtained between closely related species such as Megalobrama amblycephala and Culter alba include genome resequencing and analysis, transcriptome sequencing and analysis, etc. However, these technologies have high sequencing costs and long sequencing and analysis cycles, which are not conducive to the identification of large-scale samples. Summary of the invention The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology, provide a method for quickly and in batches identifying the genetic composition of different parental species in the hybrid offspring of closely related species such as Megalobrama amblycephala and Culter alba, and also provide a specific primer pool and application.

[0005] In order to solve the above technical problems, the technical solution proposed by the present invention is: A method for identifying the genetic composition of hybrid offspring of Megalobrama amblycephala and Culter alba, comprising the following steps: (1) Based on the existing genome data of Megalobrama amblycephala and Culter alba at the chromosome level, a specific primer pool was designed for the SNP loci; (2) Extracting genomic DNA from samples of fish material to be identified; (3) Using the genomic DNA extracted in step (2) as a template, a first round of PCR is performed by a two-step PCR method to complete the amplification of the target SNP site sequence and obtain the first round of PCR product; (4) performing a second round of PCR reaction using the first round PCR product as a template to obtain the second round PCR product; (5) Mix equal amounts of the PCR products from the second round and sequence them using a high-throughput sequencer; (6) Perform quality control on the sequencing data, perform genome alignment, SNPs identification and reads calculation on the sequencing data, and use it to determine the genotype of the hybrid individuals, and then identify the genetic composition of the hybrid offspring of the bighead carp and the black carp.

[0006] In the above method, preferably, the nucleotide sequence of the specific primer pool is as follows: 1-F: AGGGCACTAATGTCACCTGCTACA, 1-R:GCTCGGTCTTTACAACACCTGGAT; 2-F:AAAGCAATGCCTGTGTTCCGTCA, 2-R: TGTAGCAATCCCGTCCACTTCCA; 3-F:GGCTGTGTTTCCTTCTGCCTATA, 3-R: GACGAGTGCCGTTGAACTCTTCT; 4-F:GGACCTATTCCTTACCGACCA, 4-R: GCTGGCTAAAGGCTTTATCTGT; 5-F:GTGGTATGTCACCTTCCTTTTG, 5-R:CACGTCTACTATCCTCTTTGGG; 6-F:GGAACAAATACCTGAGCCTGAT, 6-R: CAGAATTCTTTAACACCCGTGAG; 7-F:GAGTTCAGGGACAACAAAGATGT, 7-R:TCTCTGAGAAGACCATTAGCACC; 8-F: CTTCAACAGAATTGGAGGCATG, 8-R:GAGGGATGAAATCTCCTTGGC; 9-F:GCTCACAGATGACCACAAACGT, 9-R:TGGCTCGAGAACAGAATGAA; 10-F:GAACCCCTCGTGCATCCAAC, 10-R:CACCGCATTCCCTCCCATCT; 11-F:TCTCTACCGCCACCACTCAAGT, 11-R:ATCCATCAAGCCATAGTTTCGA; 12-F:TCAAAGGTGAAGAACTCTGACG, 12-R:ACAGCCAATCATCTCTCCGTAT; 13-F:TAGAGGGGACAAACTCGTACTCAA, 13-R:ATCCTAAACTCACACTTTCATAA; 14-F:AGTGTGGATGCCAGTGACAAAAT, 14-R:GAGCGAATGAGTTCCCTAGACAG; 15-F:GACCTTTGCCCTAGCTCCTTTC, 15-R:GGCTGCTGTCTGTTCCATTCAC; 16-F:TCTCTGAACTTCACTCTACAAACTC, 16-R:TACGGTCTTCTCAATCTCATACTCT; 17-F:GAGGACAGCATTTTCTACACTCTG, 17-R:TCTCCATCTCCATTCAAGTCAAAC; 18-F:GAAACATATCCCAGGATTCACTCT, 18-R: CAAGGACTCAGCAACAATAACAGC; 19-F:TTTACCGTCTCCATTAGCAACAA, 19-R:GTGAACAGAGGCGAATCAAACAT; 20-F:GAACCACCCAAGAACCAAACT, 20-R:AGCGATGACATCAAACACCT; 21-F:ACATTATGTCCCATCATACGAGGTG, 21-R: TTGACTGTTAAGGAGAATCCCAGAG; 22-F:ATACAAGTTGCTCGGATGATTC, 22-R: GAAGAGGAGGAAGGTGATGAAGG; 23-F:TCACAAAGTAAACATGCTTCTGCT, 23-R:TTTCTCTTCATTTGGTGAAGTGCT; 24-F: CTCAGGAATCACTTTCTTTGGT, 24-R: TCCAGTAGGTTTGTGTTTGCCTT.

[0007] Preferably, the first round PCR system is as follows: 2 μL of 10 ng / μL DNA template; 1 μL of 10 μM upstream primer pool; 1 μL of 10 μM downstream primer pool; 15 μL of 2×PCR Ready Mix, with a total volume of 25 μL.

[0008] Preferably, the first round of PCR reaction program is as follows: pre-denaturation at 98°C for 3 min; then 8 cycles with the conditions of denaturation at 98°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 30 s; followed by 25 cycles with the conditions of denaturation at 98°C for 30 s, annealing at 66°C for 30 s, and extension at 72°C for 30 s; and finally extension at 72°C for 5 min.

[0009] Preferably, the second round PCR system is as follows: 2 μL of 10 ng / μL DNA template; 1 μL of 10 μM universal P7 primer containing molecular tags; 1 μL of 10 μM universal P5 primer; 15 μL of 2×PCR Ready Mix, with a total volume of 30 μL.

[0010] Preferably, the second round of PCR reaction program is as follows: pre-denaturation at 98°C for 5 min; then 5 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 20 s, and extension at 72°C for 30 s; finally extension at 72°C for 5 min; and insulation at 4°C after completion.

[0011] Preferably, after the first or second round of PCR reaction is completed, AMPure XP magnetic beads are used to purify and recover the PCR product.

[0012] Preferably, the PCR reaction is performed on a GeneAmp® PCR System 2700 thermal cycler, and the high-throughput sequencer is a HiSeq XTen sequencer.

[0013] Preferably, fastq quality control software is used to perform quality control on the sequencing data, and bowtie2, samtools and bedtools software are used to perform genome alignment, SNPs identification and reads calculation on the sequencing data.

[0014] Preferably, the process of determining the genotype of the hybrid individual is as follows: using specific genomic regions of Megalobrama amblycephala and Culter alba as reference sequences at the same time, using mixed samples of equal concentrations of Megalobrama F1, Megalobrama amblycephala and Culter alba DNA as controls, the base frequencies detected are used as the basis for evaluating the error range of the test results, and after filtering out sites that do not meet the credibility standards, all data results are classified and counted.

[0015] Based on a general inventive concept, the present invention also provides a specific primer pool, and the use of the specific primer pool in identifying the genetic composition of hybrid offspring of Megalobrama amblycephala and Culter alba.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention innovatively uses a method combining multiplex PCR and high-throughput sequencing to qualitatively and quantitatively detect the base conditions of SNP sites on each pair of homologous chromosomes of Megalobrama amblycephala and Culter alba, thereby providing an efficient identification technology for the genetic composition of hybrid offspring obtained between other closely related species such as Megalobrama amblycephala and Culter alba. Compared with whole genome sequencing genes, this method only amplifies and sequences specific regions on each chromosome, which effectively reduces sequencing costs and reduces analysis cycles. It is an efficient technology for quickly identifying the genetic composition of hybrid offspring obtained by hybridization of closely related species such as Megalobrama amblycephala and Culter alba. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 It is the display of offline sequencing data results; the first line is the name of a sequencing read, the second line is the sequencing base information, the third line is the specific symbol, and the fourth line is the quality symbol corresponding to the sequencing base in the second line.

[0019] Figure 2It is a schematic diagram of the analysis results; Fragment name: the name of the target region; Length: the length of the specific region; Pos: the position of the specific region; Chr: the corresponding chromosome number; Start: the starting position; End: the ending position; Depth: the sequencing depth of the region; Genotype: genotype; Ref: the reference base; Alt: the check base in the sample; Alt_depth: the sequencing depth of the mutant base; Alt_ratio: the mutation ratio of the site in the sample; Error_ratio: the percentage of sequencing errors. DETAILED DESCRIPTION

[0020] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the following specific embodiments.

[0021] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0022] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0023] The present invention utilizes the genome data of Megalobrama amblycephala and Culter alba at the existing chromosome level to screen the orthologous chromosome pairs between the species of Megalobrama amblycephala and Culter alba, and finally uses a large number of SNPs in the homologous regions on 24 pairs of homologous chromosomes from Megalobrama amblycephala and Culter alba as identification markers, and screens highly conservative coding regions on each pair of chromosomes to design a multiplex PCR primer pool, and obtains sequencing reads data of a specific genomic region through multiplex PCR and high-throughput sequencing, and evaluates species-specific SNPs on the genomic region by comparing with a reference genome, and calculates the ratio of reads from Megalobrama amblycephala and Culter alba, and uses the ratio to determine the genotype of each pair of homologous chromosomes of the hybrid individuals.

[0024] Specifically, the above method can be performed as follows: (1) Based on the known chromosome-mounted genome data of Megalobrama amblycephala and Culter alba, a specific primer pool was designed for the SNP loci; (2) Extracting genomic DNA from samples of fish material to be identified; (3) Using the genomic DNA extracted in step (2) as a template, a two-step PCR method was used to amplify the target SNP site sequence and prepare a compatible Illumina sequencing library. The first round of PCR system is as follows: DNA template (10 ng / μL) 2μL; upstream primer pool (10 μM) 1μL; downstream primer pool (10 μM) 1μL; 2×PCR Ready Mix 15 μL (total volume 25μL) (Kapa HiFi Ready Mix). After the PCR reaction is completed, the PCR product is detected by 1% agarose gel electrophoresis to confirm that the product size is correct, and the PCR product is purified and recovered using AMPure XP magnetic beads.

[0025] (4) Perform the second round of PCR reaction using the first round PCR product as a template. The reaction system is as follows: DNA template (10 ng / μL) 2μL, universal P7 primer (containing molecular tag, 10μM) 1μL; universal P5 primer (10 μM) 1μL; 2×PCR ReadyMix 15μL (total volume 30μL). To obtain a library with molecular tags for sequencing.

[0026] (5) Sequence the second-round PCR products using a high-throughput sequencer.

[0027] (6) Use quality control software such as fastq to perform quality control on sequencing data. Use software such as bowtie2, samtools, and bedtools to perform genome alignment, SNPs identification, and reads calculation on sequencing data, and ultimately use them to determine the genotype of hybrid individuals.

[0028] Embodiment 1: 1. Prepare experimental materials Megalobrama amblycephala (2n=48) and Culter alburnus (2n=48), as well as their hybridization results in the F1 (Megalobrama F1, 2n=48) and F2 (Megalobrama F2, 2n=48).

[0029] 2. Extraction of genomic DNA For the adults of the prepared F1 and F2, blood (or part of the fin rays) was collected from the vein of each fish material sample using a disposable syringe, and genomic DNA was extracted using the UNIQ-10 column-based genomic DNA extraction kit (Shanghai Sangon Company). The concentration of the extracted DNA was detected using an ultraviolet spectrophotometer and stored at -20°C.

[0030] 3. Primer Design Combining the genomic data of Megalobrama amblycephala and Culter alba which have been mapped to the chromosome level and the transcriptome data of their hybrid offspring, the visualization tool IGV software was used to browse and analyze the sequencing data, and a coding sequence (CDS) region with more single nucleotide polymorphism (SNP) sites on each pair of chromosomes was screened for primer design (Table 1).

[0031] Table 1. Primer sequences for species-specific fragment amplification (shown in SEQ ID No. 1-48)

[0032] 4. Multiplex PCR Amplification The genomic DNA of each fish material sample extracted in step 2 was used as a template, and the primers obtained in step 3 were used as the reaction primer pool to design a PCR system for amplification to obtain the amplified product. The PCR reaction was carried out on a GeneAmp® PCR System2700 thermal cycler (produced by Applied Biosystems, USA). The total volume of the first round of PCR amplification reaction was 25 μL, including 2 μL of DNA template (10 ng / μL); 1 μL of each upstream and downstream primer pool (10 μM); and 15 μL of 2×PCR Ready Mix (Kapa HiFi Ready Mix). The PCR reaction program was as follows: 98 °C pre-denaturation for 3 min; then 8 cycles of 98 °C denaturation for 30 s, 50 °C annealing for 30 s, and 72 °C extension for 30 s; followed by 25 cycles of 98 °C denaturation for 30 s, 66 °C annealing for 30 s, and 72 °C extension for 30 s; and finally 72 °C extension for 5 min. After the PCR reaction was completed, the PCR product was detected by 1% agarose gel electrophoresis. After confirming that the product size was correct, the PCR product was purified and recovered using AMPure XP magnetic beads. Then, the second round of PCR reaction was performed using the first round PCR product as a template. The total reaction volume was 30 μL, including 2 μL of DNA template (10 ng / μL), 1 μL of universal P7 primer (containing molecular tag, 10 μM); 1 μL of universal P5 primer (10 μM); and 15 μL of 2×PCRReady Mix (total volume 30 μL). The following PCR program was performed: pre-denaturation at 98 °C for 5 min; then 5 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 20 s, and extension at 72 °C for 30 s; and finally extension at 72 °C for 5 min. After completion, the cells were incubated at 4 °C. The final PCR product was purified and recovered using AMPure XP magnetic beads.

[0033] 5. High-throughput sequencing and data analysis After equal amounts of PCR products were mixed, they were sequenced using a HiSeq XTen sequencer (Illumina, San Diego, CA) (other high-throughput sequencing technologies can also be used for analysis), see Figure 1 . Finally, the data were subjected to quality control and genotyping analysis. The specific genomic regions of Megalobrama amblycephala and Culter alba were used as reference sequences at the same time, and the base frequencies detected by the control (Grilled Broiler F1, mixed samples of equal concentrations of Megalobrama amblycephala and Culter alba DNA) were used as the basis for evaluating the error range of the test results. After filtering out sites with poor credibility, all data results were classified and counted. The statistical results of the SNP site detection of Megalobrama F2 obtained in this example are shown in Table 2. Some analysis results are shown in Table 2. Figure 2 .

[0034] Table 2: Statistics of SNP loci detection results in the F2 population of bream

[0035] Note: Alt=50±10%: the ratio of reads aligned between homologous regions of Megalobrama amblycephala and Culter alba is within the range of 50±10%; Alt=100%: the ratio of reads aligned between homologous regions of Megalobrama amblycephala and Culter alba is 100%; Alt=0%: the ratio of reads aligned between homologous regions of Megalobrama amblycephala and Culter alba is 0%; Alt≠(0, 50±10%, 100%): other types of situations.

[0036] As can be seen from the above, in this embodiment, based on the genome data of Megalobrama amblycephala and Culter alba mounted at the chromosome level, the CDS region sequences with more SNP sites on each pair of homologous chromosomes are screened, and the genetic composition of hybrid fish is identified by using a method combining multiple PCR and high-throughput sequencing, with each pair of homologous chromosomes as the research unit. The results show that this method can identify the proportion of Megalobrama amblycephala type bases and Culter alba type bases at each pair of chromosome SNP sites in the hybrid individuals, thereby quantitatively analyzing the genetic composition of different hybrid individuals. The molecular marker technology based on the SNP sites on each pair of homologous chromosome genomes of Megalobrama amblycephala and Culter alba can quickly, accurately and specifically identify the genetic composition of the hybrid offspring of Megalobrama amblycephala and Culter alba.

Claims

1. A method for identifying the genetic composition of hybrid offspring of Megalobrama amblycephala and Culter alba, characterized in that: The steps include: (1) Based on the existing genome data of Megalobrama amblycephala and Culter alba at the chromosome level, a specific primer pool was designed for the SNP loci; (2) Extracting genomic DNA from samples of fish material to be identified; (3) Using the genomic DNA extracted in step (2) as a template, a first round of PCR is performed by a two-step PCR method to complete the amplification of the target SNP site sequence and obtain the first round of PCR product; (4) performing a second round of PCR reaction using the first round PCR product as a template to obtain the second round PCR product; (5) Mixing equal amounts of the PCR products from the second round and sequencing them using a high-throughput sequencer; (6) Perform quality control on the sequencing data, perform genome alignment, SNPs identification and reads calculation on the sequencing data, determine the genotype of the hybrid individuals, and then identify the genetic composition of the hybrid offspring of the bighead carp and the black carp.

2. The method according to claim 1, characterized in that The specific primer pool contains 24 pairs of primers, and the nucleotide sequences are as follows: 1-F: AGGGCACTAATGTCACCTGCTACA, 1-R:GCTCGGTCTTTACAACACCTGGAT; 2-F:AAAGCAATGCCTGTGTTCCGTCA, 2-R: TGTAGCAATCCCGTCCACTTCCA; 3-F:GGCTGTGTTTCCTTCTGCCTATA, 3-R: GACGAGTGCCGTTGAACTCTTCT; 4-F:GGACCTATTCCTTACCGACCA, 4-R: GCTGGCTAAAGGCTTTATCTGT; 5-F:GTGGTATGTCACCTTCCTTTTG, 5-R:CACGTCTACTATCCTCTTTGGG; 6-F:GGAACAAATACCTGAGCCTGAT, 6-R: CAGAATTCTTTAACACCCGTGAG; 7-F:GAGTTCAGGGACAACAAAGATGT, 7-R:TCTCTGAGAAGACCATTAGCACC; 8-F: CTTCAACAGAATTGGAGGCATG, 8-R:GAGGGATGAAATCTCCTTGGC; 9-F:GCTCACAGATGACCACAAACGT, 9-R:TGGCTCCAGAACAGAGAATGAA; 10-F:GAACCCCTCGTGCATCCAAC, 10-R:CACCGCATTCCCTCCCATCT; 11-F: TTCCTACCGCCACAACCTCAAGT, 11-R:ATCCATCAAGCCATAGTTTCGA; 12-F:TCAAAGGTGAAGAACTCTGACG, 12-R:ACAGCCAATCATCTCTCCGTAT; 13-F:TAGAGGGACAAACTCGTACTCAA, 13-R: ATCCTAAACTCACACTTTCATAA; 14-F: AGTGTGGATGCCAGTGACAAAAT, 14-R:GAGCGAATGAGTTCCCTAGACAG; 15-F: GACCTTTGCCCTAGCTCCTTTC, 15-R:GGCTGCTGTCTGTTCCATTCAC; 16-F: TCTCTGAACTTCACTCTACAAACTC, 16-R: TACGGTCTTCTCAATCTCATACTCT; 17-F:GAGGACAGCATTTTCTACACTCTG, 17-R:TCTCCATCTCCATTCAAGTCAAAC; 18-F: GAAACATATCCCAGGATTCACTCT, 18-R: CAAGGACTCAGCAACAATAACAGC; 19-F: TTTACCGTCTCCATAGCAACAA, 19-R:GTGAACAGAGGCGAATCAAACAT; 20-F:GAACCACCCAAGACCAAACT, 20-R:AGCGATGACATCAAACACCT; 21-F: ACATTATGTCCCATCATACGAGGTG, 21-R: TTGACTGTTAAGGAGAATCCCAGAG; 22-F:ATACAAGTTGCTCGGATGATTC, 22-R: GAAGAGGAGGAAGGTGATGAAGG; 23-F:TCACAAAGTAAACATGCTTCTGCT, 23-R:TTTCTCTTCATTTGGTGAAGTGCT; 24-F: CTCAGGAATCACTTTCTTTGGT, 24-R: TCCAGTAGGTTTGTGTTTGCCTT.

3. The method according to claim 1, characterized in that The first-round PCR system was as follows: 2 μL of 10 ng / μL DNA template; 1 μL of 10 μM upstream primer pool; 1 μL of 10 μM downstream primer pool; 15 μL of 2×PCR Ready Mix, with a total volume of 25 μL; the first-round PCR reaction program was as follows: pre-denaturation at 98 °C for 3 min; then 8 cycles were performed with the conditions of denaturation at 98 °C for 30 s, annealing at 50 °C for 30 s, and extension at 72 °C for 30 s; followed by 25 cycles with the conditions of denaturation at 98 °C for 30 s, annealing at 66 °C for 30 s, and extension at 72 °C for 30 s; and finally extension at 72 °C for 5 min.

4. The method according to claim 1, characterized in that The second-round PCR system was as follows: 2 μL of 10 ng / μL DNA template; 1 μL of 10 μM universal P7 primer containing molecular tags; 1 μL of 10 μM universal P5 primer; 15 μL of 2×PCR Ready Mix, with a total volume of 30 μL; the second-round PCR reaction program was as follows: pre-denaturation at 98 °C for 5 min; then 5 cycles were performed, with the conditions of denaturation at 94 °C for 30 s, annealing at 55 °C for 20 s, and extension at 72 °C for 30 s; finally extension at 72 °C for 5 min; and insulation at 4 °C after completion.

5. The method according to claim 1, characterized in that After the first or second round of PCR reaction, the PCR products were purified and recovered using AMPure XP magnetic beads.

6. The method according to claim 1, characterized in that PCR reactions were performed on a GeneAmp® PCR System 2700 thermal cycler.

7. The method according to claim 1, characterized in that Fastq quality control software was used to control the sequencing data, and bowtie2, samtools and bedtools software were used to perform genome alignment, SNPs identification and reads calculation on the sequencing data.

8. The method according to any one of claims 1 to 8, characterized in that The process of determining the genotype of hybrid individuals is as follows: specific genomic regions of Megalobrama amblycephala and Culter alba are used as reference sequences at the same time, and mixed samples of equal concentrations of Megalobrama amblycephala and Culter alba DNA are used as controls. The base frequencies detected are used as the basis for evaluating the error range of the detection results. After filtering out sites that do not meet the credibility standards, all data results are classified and counted.

9. A specific primer pool, characterized in that: The specific primer pool contains 24 pairs of primers, and the nucleotide sequences are as follows: 1-F: AGGGCACTAATGTCACCTGCTACA, 1-R:GCTCGGTCTTTACAACACCTGGAT; 2-F:AAAGCAATGCCTGTGTTCCGTCA, 2-R: TGTAGCAATCCCGTCCACTTCCA; 3-F:GGCTGTGTTTCCTTCTGCCTATA, 3-R: GACGAGTGCCGTTGAACTCTTCT; 4-F:GGACCTATTCCTTACCGACCA, 4-R: GCTGGCTAAAGGCTTTATCTGT; 5-F:GTGGTATGTCACCTTCCTTTTG, 5-R:CACGTCTACTATCCTCTTTGGG; 6-F:GGAACAAATACCTGAGCCTGAT, 6-R: CAGAATTCTTTAACACCCGTGAG; 7-F:GAGTTCAGGGACAACAAAGATGT, 7-R:TCTCTGAGAAGACCATTAGCACC; 8-F: CTTCAACAGAATTGGAGGCATG, 8-R:GAGGGATGAAATCTCCTTGGC; 9-F:GCTCACAGATGACCACAAACGT, 9-R:TGGCTCCAGAACAGAGAATGAA; 10-F:GAACCCCTCGTGCATCCAAC, 10-R:CACCGCATTCCCTCCCATCT; 11-F: TTCCTACCGCCACAACCTCAAGT, 11-R:ATCCATCAAGCCATAGTTTCGA; 12-F:TCAAAGGTGAAGAACTCTGACG, 12-R:ACAGCCAATCATCTCTCCGTAT; 13-F:TAGAGGGACAAACTCGTACTCAA, 13-R: ATCCTAAACTCACACTTTCATAA; 14-F: AGTGTGGATGCCAGTGACAAAAT, 14-R:GAGCGAATGAGTTCCCTAGACAG; 15-F: GACCTTTGCCCTAGCTCCTTTC, 15-R:GGCTGCTGTCTGTTCCATTCAC; 16-F: TCTCTGAACTTCACTCTACAAACTC, 16-R: TACGGTCTTCTCAATCTCATACTCT; 17-F:GAGGACAGCATTTTCTACACTCTG, 17-R:TCTCCATCTCCATTCAAGTCAAAC; 18-F: GAAACATATCCCAGGATTCACTCT, 18-R: CAAGGACTCAGCAACAATAACAGC; 19-F: TTTACCGTCTCCATAGCAACAA, 19-R:GTGAACAGAGGCGAATCAAACAT; 20-F:GAACCACCCAAGACCAAACT, 20-R:AGCGATGACATCAAACACCT; 21-F: ACATTATGTCCCATCATACGAGGTG, 21-R: TTGACTGTTAAGGAGAATCCCAGAG; 22-F:ATACAAGTTGCTCGGATGATTC, 22-R: GAAGAGGAGGAAGGTGATGAAGG; 23-F:TCACAAAGTAAACATGCTTCTGCT, 23-R:TTTCTCTTCATTTGGTGAAGTGCT; 24-F: CTCAGGAATCACTTTCTTTGGT, 24-R: TCCAGTAGGTTTGTGTTTGCCTT.

10. Use of the specific primer pool according to claim 9 in identifying the genetic composition of hybrid offspring of Megalobrama amblycephala and Culter alba.

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