Method for rapidly identifying genetic composition of hybrid progeny of mandarin fish and topmouth gudgeon, specific primer pool and application

Abstract

The present application belongs to the field of molecular biology and animal molecular breeding, and discloses a method for identifying genetic composition of hybrid offspring of Misgurnus anguillicaudatus and Culter alburnus, a specific primer pool, and application of the specific primer pool in identifying genetic composition of hybrid offspring of Misgurnus anguillicaudatus and Culter alburnus. The present application innovatively uses a method combining multiplex PCR and high-throughput sequencing to qualitatively and quantitatively detect base conditions of SNP sites on each pair of homologous chromosomes of Misgurnus anguillicaudatus and Culter alburnus, thereby providing an efficient identification technology for genetic composition of hybrid offspring obtained between Misgurnus anguillicaudatus and other species of close relatives such as Culter alburnus. Compared with whole genome sequencing genes, the method only amplifies and sequences specific regions on each chromosome, which effectively reduces sequencing cost and analysis period, and is an efficient technology for rapidly identifying genetic composition of hybrid offspring obtained by hybridization of Misgurnus anguillicaudatus and other close relatives such as Culter alburnus.

Description

TECHNICAL FIELD

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

[0002] Megalobrama amblycephala is a unique fish in the middle-sized lakes of the middle and lower reaches of the Yangtze River in China. Since the success of artificial breeding technology in 1964, Megalobrama amblycephala has become the main force of freshwater aquaculture in China. This fish is favored by breeders due to its wide food habits, low breeding cost, fast growth rate, high survival rate, easy catching and easy breeding characteristics. The annual output of Megalobrama amblycephala is stable at 700 to 800 tons, and it is a herbivorous fish, known for its high back and short body shape. Culter alburnus 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, Culter alburnus has become an indispensable part of freshwater aquaculture. This fish mainly feeds on plankton and belongs to carnivorous fish.

[0003] By crossing Megalobrama amblycephala and Culter alburnus as parents, new breed lines with bisexual fertility can be successfully constructed. Using the F1 generation of these new breed lines and the original parents, fish breeds with significant hybrid advantages can be bred. For example, hybrid Culter alburnus (Aquatic New Breed Certificate Registration No.: GS-02-003-2014), Heifang crucian carp, Xiangjun bream, etc. These breeds not only inherit the excellent traits of herbivory, fast growth, low oxygen tolerance and tender meat, but also have great market potential.

[0004] Currently, the techniques for identifying the genotype of hybrid offspring obtained from species with close genetic relationship such as Megalobrama amblycephala and Culter alburnus include genome resequencing and analysis, transcriptome sequencing and analysis, etc. However, these techniques have high sequencing cost, long sequencing and analysis period, etc., which are not conducive to large-scale sample identification work. SUMMARY

[0005] The technical problem to be solved by the present application is to overcome the deficiencies and defects mentioned in the above background technology, and to provide a method for quickly and batch identifying the genetic composition of different parent species in the hybrid offspring of Megalobrama amblycephala and Culter alburnus and other closely related species, as well as a specific primer pool and application.

[0006] To solve the above technical problems, the technical solution proposed by the present application is as follows:

[0007] A method for identifying the genetic composition of hybrid offspring of Megalobrama amblycephala and Culter alburnus, comprising the following steps:

[0008] (1) According to the existing genome data of Megalobrama amblycephala and Culter alburnus at the chromosome level, a specific primer pool is designed for SNP sites;

[0009] (2) extracting genomic DNA of the sample of fish material to be identified;

[0010] (3) taking the genomic DNA extracted in step (2) as a template, then performing first-round PCR by a two-step PCR method to complete amplification of the sequence of the target SNP site, and obtaining first-round PCR products;

[0011] (4) taking the first-round PCR products as a template to perform second-round PCR reaction, and obtaining second-round PCR products;

[0012] (5) mixing the second-round PCR products in equal amounts, and then using a high-throughput sequencer to perform sequencing;

[0013] (6) performing quality control on the sequencing data, performing genomic alignment, SNP identification and read calculation on the sequencing data, and using the sequencing data to determine the genotype of the hybrid individual, and then identifying the genetic composition of the offspring of the hybrid between Misgurnus anguillicaudatus and Culter alburnus.

[0014] In the above method, preferably, the nucleotide sequences of the specific primer pool are as follows:

[0015] 1-F: AGGGCACTAATGTCACCTGCTACA,

[0016] 1-R: GCTCGGTCTTTACAACACCTGGAT;

[0017] 2-F: AAAGCAATGCCTGTGTTCCGTCA,

[0018] 2-R: TGTAGCAATCCCGTCCACTTCCA;

[0019] 3-F: GGCTGTGTTTCCTTCTGCCTATA,

[0020] 3-R: GACGAGTGCCGTTGAACTCTTCT;

[0021] 4-F: GGACCTATTCCTTTACCGACCA,

[0022] 4-R: GCTGGCTAAAGGCTTTATCTGT;

[0023] 5-F: GTGGTATGTCACCTTCCTTTTG,

[0024] 5-R: CACGTCTACTATCCTCTTTGGG;

[0025] 6-F: GGAACAAATACCTGAGCCTGAT,

[0026] 6-R: CAGAATCTTTAACACCCGTGAG;

[0027] 7-F: GAGTTCAGGGACAACAAAGATGT,

[0028] 7-R: TCTCTGAGAAGACCATTAGCACC;

[0029] 8-F: CTTCAACAGAATTGGAGGCATG,

[0030] 8-R: GAGGGATGAAATCTTCCTTGGC;

[0031] 9-F: GCTCACAGATGACCACAAACGT,

[0032] 9-R: TGGCTCCAGAACAGAGAATGAA;

[0033] 10-F: GAACCCCTCGTGCATCCAAC,

[0034] 10-R: CACCGCATTCCCTCCCATCT;

[0035] 11-F: TCTCTACCGCCACACCTCAAGT,

[0036] 11-R: ATCCATCAAGCCATAGTTTCGA;

[0037] 12-F: TCAAAGGTGAAGAACTCTGACG,

[0038] 12-R: ACAGCCAATCATCTCTCCGTAT;

[0039] 13-F: TAGAGGGACAAACTCGTACTCAA,

[0040] 13-R: ATCCTAAACTCACACTTTCATAA;

[0041] 14-F: AGTGTGGATGCCAGTGACAAAAT,

[0042] 14-R: GAGCGAATGAGTTCCCTAGACAG;

[0043] 15-F: GACCTTTGCCCTAGCTCCTTTC,

[0044] 15-R: GGCTGCTGTCTGTTCCATTCAC;

[0045] 16-F: TCTCTGAACTTCACTCTACAAACTC,

[0046] 16-R: TACGGTCTTCTCAATCTCATACTCT;

[0047] 17-F: GAGGACAGCATTTTCTACACTCTG,

[0048] 17-R: TCTCCATCTCCATTCAAGTCAAAC;

[0049] 18-F: GAAACATATCCCAGGATTCACTCT,

[0050] 18-R: CAAGGACTCAGCAACAATAACAGC;

[0051] 19-F: TTTACCGTCTCCATTAGCAACAA,

[0052] 19-R: GTGAACAGAGGCGAATCAAACAT;

[0053] 20-F: GAACCACCCAAGACCAAACT,

[0054] 20-R: AGCGATGACATCAAACACCT;

[0055] 21-F: ACATTATGTCCCATCATACGAGGTG,

[0056] 21-R: TTGACTGTTAAGGAGAATCCCAGAG;

[0057] 22-F: ATACAAGTTGCTGCGGATGATTC,

[0058] 22-R: GAAGAGGAGGAAGGTGATGAAGG;

[0059] 23-F: TCACAAAGTAAACATGCTTCTGCT,

[0060] 23-R: TTTCTCTTCATTTGGTGAAGTGCT;

[0061] 24-F: CTCAGGAATCACTTTCTTTGGT,

[0062] 24-R: TCCAGTAGGTTTGTGTTTGCCTT.

[0063] Preferably, the first round of PCR system is as follows: 10 ng / μL DNA template 2 μL; 10 μM upstream primer pool 1 μL; 10 μM downstream primer pool 1 μL; 2x PCR Ready Mix 15 μL, total volume 25 μL.

[0064] Preferably, the first round of PCR reaction program is as follows: 98 ℃ pre-denaturation 3 min; then perform 8 cycles, condition is 98 ℃ denaturation 30 s, 50 ℃ annealing 30 s, 72 ℃ extension 30 s; followed by 25 cycles, condition is 98 ℃ denaturation 30 s, 66 ℃ annealing 30 s, 72 ℃ extension 30 s; finally 72 ℃ extension 5 min.

[0065] Preferably, the second round of PCR system is as follows: 10 ng / μL DNA template 2 μL; 10 μM molecular tag containing universal P7 primer 1 μL; 10 μM universal P5 primer 1 μL; 2x PCR Ready Mix 15 μL, total volume 30 μL.

[0066] Preferably, the second round of PCR reaction program is as follows: 98 ℃ pre-denaturation 5 min; then perform 5 cycles, condition is denaturation 94 ℃ 30 s, 55 ℃ annealing 20 s, 72 ℃ extension 30 s; finally 72 ℃ extension 5 min; after completion, 4 ℃ incubation.

[0067] Preferably, after the first or second round of PCR reaction is completed, the PCR product is purified and recovered using AMPure XP magnetic beads.

[0068] Preferably, the PCR reaction is performed on a GeneAmp® PCR System 2700 thermal cycler, and the high-throughput sequencer is a HiSeq XTen sequencer.

[0069] Preferably, the sequencing data is quality controlled using fastq quality control software, and the sequencing data is genome aligned, SNPs identified and reads calculated using bowtie2, samtools and bedtools software.

[0070] Preferably, the process of judging the genotype of the hybrid individual is as follows: the specific genomic region of the mandarin fish and the topmouth culter is simultaneously used as a reference sequence, the DNA of the mandarin fish and the topmouth culter mixed at the same concentration is used as a control to detect the base frequency, and after filtering out sites with insufficient credibility, all data results are classified and counted.

[0071] Based on a total inventive concept, the application also provides a specific primer pool and application of the specific primer pool in identifying genetic composition of hybrid offspring of Megalobrama amblycephala and Procyprinus.

[0072] Compared with the prior art, the application has the following beneficial effects:

[0073] The application innovatively uses a method combining multiplex PCR and high-throughput sequencing to qualitatively and quantitatively detect the base condition of SNP sites on each pair of homologous chromosomes of Megalobrama amblycephala and Procyprinus, thereby providing a high-efficiency identification technology for genetic composition of hybrid offspring obtained by hybridization between Megalobrama amblycephala and other species close thereto, such as Procyprinus. Compared with whole genome sequencing genes, the method only amplifies and sequences specific regions on each chromosome, which effectively reduces the sequencing cost and also reduces the analysis period, and is a high-efficiency technology for rapidly identifying genetic composition of hybrid offspring obtained by hybridization between Megalobrama amblycephala and other species close thereto, such as Procyprinus. BRIEF DESCRIPTION OF DRAWINGS

[0074] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0075] Figure 1 is a result display of sequencing data; the first line is the name of a sequencing read, the second line is the sequencing base information, the third line is a specific symbol, and the fourth line is the quality symbol corresponding to the second line of sequencing base.

[0076] Figure 2 is a schematic diagram of analysis results; Fragment name: naming of the target region; Length: length of the specific region; Pos: position of the specific region; Chr: corresponding chromosome number; Start: starting position; End: end position; Depth: sequencing depth of the region; Genotype: genotype; Ref: reference base; Alt: checking base in the sample; Alt_depth: sequencing depth of the mutant base; Alt_ratio: mutation ratio of the sample at the site; Error_ratio: sequencing error percentage. DETAILED DESCRIPTION

[0077] In order to facilitate the understanding of the application, the following will combine the drawings in the specification and the preferred embodiments to make a more comprehensive and detailed description of the application, but the protection scope of the application is not limited to the following specific embodiments.

[0078] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application.

[0079] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present application can be purchased on the market or can be prepared by existing methods.

[0080] The present application utilizes the existing chromosome level genome data of the Megalobrama amblycephala and Culter alburnus, screens the orthologous chromosome pairs between the Megalobrama amblycephala and Culter alburnus species, and finally uses a large number of SNPs on the homologous regions of 24 pairs of homologous chromosomes of the Megalobrama amblycephala and Culter alburnus as identification markers, and screens the coding regions with high conservation on each pair of chromosomes to carry out the design of multiplex PCR primer pool. Through multiplex PCR and high-throughput sequencing, sequencing reads data of specific genomic regions can be obtained, and through alignment with the reference genome, species-specific SNPs on the genomic region can be evaluated, and the proportion of Megalobrama amblycephala and Culter alburnus derived reads is calculated, and is used to judge the genotype of each pair of homologous chromosomes of the hybrid individual.

[0081] Specifically, the above method can be operated as follows:

[0082] (1) According to the known Megalobrama amblycephala and Culter alburnus genome data of the mounted chromosomes, specific primer pools are designed for SNP sites;

[0083] (2) Extracting the genomic DNA of the fish material sample to be identified;

[0084] (3) Using the genomic DNA extracted in the above step (2) as a template, then the amplification of the target SNP site sequence and the preparation of the compatible Illumina sequencing library are completed by the two-step PCR method. The first round of PCR system is as follows: DNA template (10 ng / μL) 2 μL; upstream primer pool (10 μM) 1 μL; downstream primer pool (10 μM) 1 μL; 2×PCR Ready Mix 15 μL (total volume 25 μL) (Kapa HiFi Ready Mix). After the completion of the PCR reaction, the PCR product is detected by 1% agarose gel electrophoresis to determine that the product size is correct, and the PCR product is purified and recovered by using AMPure XP magnetic beads.

[0085] (4) Using the first-round PCR product as a template, perform a second-round PCR reaction. The reaction system is as follows: DNA template (10 ng / μL) 2 μL, universal P7 primer (containing molecular tag, 10 μM) 1 μL; universal P5 primer (10 μM) 1 μL; 2×PCR ReadyMix 15 μL (total volume 30 μL). This is to obtain a sequencing library with a molecular tag.

[0086] (5) Sequencing of the second round of PCR products using a high-throughput sequencer.

[0087] (6) Use quality control software such as fastq to perform quality control on sequencing data. Software such as bowtie2, samtools and bedtools can be used to perform genome alignment, SNP identification and read calculation on sequencing data, and finally use them to determine the genotype of hybrid individuals.

[0088] Example 1:

[0089] 1. Prepare experimental materials

[0090] The blunt snout bream (Megalobrama amblycephala, 2n=48) and the topmouth bream (Culter alburnus, 2n=48), as well as their hybrid F1 (Culter alburnus F1, 2n=48) and F2 (Culter alburnus F2, 2n=48).

[0091] 2. Extraction of genomic DNA

[0092] For the prepared adult Culter alburnus F1 and F2, blood (or partial fin rays) was collected from the veins of each fish 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 measured using a UV spectrophotometer and stored at -20℃.

[0093] 3. Primer design

[0094] Combining the genome data of blunt snout bream and topmouth bream that have been mapped to the chromosome level with the transcriptome data of their hybrid offspring, the sequencing data were browsed and analyzed using the visualization tool IGV software. A coding sequence (CDS) region with a large number of single nucleotide polymorphism (SNP) sites was selected on each pair of chromosomes for primer design (Table 1).

[0095] Table 1. Primer sequences for species-specific fragment amplification (as shown in SEQ ID No. 1-48 in sequence)

[0096]

[0097] 4. Multiplex PCR amplification

[0098] The genomic DNA of each fish material sample extracted in Step 2 above was used as a template, and the primers obtained in Step 3 were used as a reaction primer pool to design a PCR system for amplification to obtain an amplification product. The PCR reaction was performed on a GeneAmp® PCR System 2700 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 of the upper and lower primer pools (10 μM); 15 μL of 2x PCR Ready Mix (Kapa HiFi Ready Mix). The PCR reaction program was as follows: pre-denaturation at 98 ℃ for 3 min; then 8 cycles of denaturation at 98 ℃ for 30 s, annealing at 50 ℃ for 30 s, and extension at 72 ℃ for 30 s; followed by 25 cycles of denaturation at 98 ℃ for 30 s, annealing at 66 ℃ for 30 s, and extension at 72 ℃ for 30 s; and finally extension at 72 ℃ for 5 min. After the completion of the PCR reaction, the PCR product was detected by 1% agarose gel electrophoresis, and after the correct size of the product was confirmed, the PCR product was purified and recovered using AMPure XP magnetic beads. Then, the first round of PCR product was used as a template to perform a second round of PCR reaction, with a total reaction volume of 30 μL, including 2 μL of DNA template (10 ng / μL), 1 μL of universal P7 primer (containing a molecular tag, 10 μM), 1 μL of universal P5 primer (10 μM), and 15 μL of 2x PCR Ready Mix (total volume 30 μL). The following PCR program was performed: pre-denaturation at 98 ℃ for 5 min; then 5 cycles of denaturation at 94 ℃ for 30 s, annealing at 55 ℃ for 20 s, and extension at 72 ℃ for 30 s; and finally extension at 72 ℃ for 5 min. After completion, the temperature was kept at 4 ℃. The final PCR product was purified and recovered using AMPure XP magnetic beads.

[0099] 5. High-throughput sequencing and data analysis

[0100] After equal mixing of each PCR product, sequencing was performed using a HiSeq XTen sequencer (Illumina, San Diego, CA) (other high-throughput sequencing techniques can also be used for analysis), as shown in Figure 1Finally, the data is quality controlled and genotyped. The specific genomic region of Megalobrama amblycephala and Culter alburnus is used as a reference sequence, and the base frequency detected by the control (Megalobrama amblycephala and Culter alburnus DNA mixed sample with the same concentration) is used as the basis for evaluating the error range of the detection result. After filtering out the sites with poor reliability, the statistical results of all data are classified. The statistical results of the SNP site detection of the Megalobrama amblycephala and Culter alburnus F2 obtained in this embodiment are shown in Table 2. Some analysis results are shown in Figure 2 .

[0101] Table 2: Statistical results of SNP site detection in Megalobrama amblycephala and Culter alburnus F2 population

[0102]

[0103] Note: Alt = 50 ± 10%: The proportion of reads in the homologous region of Megalobrama amblycephala and Culter alburnus is in the range of 50 ± 10%; Alt = 100%: The proportion of reads in the homologous region of Megalobrama amblycephala and Culter alburnus is 100%; Alt = 0%: The proportion of reads in the homologous region of Megalobrama amblycephala and Culter alburnus is 0%; Alt ≠ (0, 50 ± 10%, 100%): Other types of conditions.

[0104] As can be seen from the above, in this embodiment, according to the genomic data of Megalobrama amblycephala and Culter alburnus mounted to the chromosome level, the CDS region sequence with more SNP sites on each pair of homologous chromosomes is screened, and the method of multiplex PCR combined with high-throughput sequencing is applied to identify the genetic composition of hybrid fish as a research unit. The results show that this method can identify the proportion of Megalobrama amblycephala type base and Culter alburnus type base on each pair of chromosomes in hybrid individuals, so as to quantitatively analyze the genetic composition of different hybrid individuals. The molecular marker technology based on the SNP site on each pair of homologous chromosomes of Megalobrama amblycephala and Culter alburnus can quickly, accurately and specifically identify the genetic composition of the hybrid offspring of Megalobrama amblycephala and Culter alburnus.

Claims

1. A method for identifying the genetic composition of the progeny of a hybrid between a Megalobrama amblycephala and a Procyprinus summerae, characterized in that, Comprising the following steps: (1) extracting genomic DNA of fish material sample to be identified; (2) taking the genomic DNA extracted in step (1) as a template, then performing first round PCR with specific primer pool to complete amplification of target SNP site sequence, and obtaining first round PCR product; The specific primer pool comprises 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: GGACCTATTCCTTTACCGACCA, 4-R: GCTGGCTAAAGGCTTTATCTGT; 5-F: GTGGTATGTCACCTTCCTTTTG, 5-R: CACGTCTACTATCCTCTTTGGG; 6-F: GGAACAAATACCTGAGCCTGAT, 6-R: CAGAATCTTTAACACCCGTGAG; 7-F: GAGTTCAGGGACAACAAAGATGT, 7-R: TCTCTGAGAAGACCATTAGCACC; 8-F: CTTCAACAGAATTGGAGGCATG, 8-R: GAGGGATGAAATCTTCCTTGGC; 9-F: GCTCACAGATGACCACAAACGT, 9-R: TGGCTCCAGAACAGAGAATGAA; 10-F: GAACCCCTCGTGCATCCAAC, 10-R: CACCGCATTCCCTCCCATCT; 11-F: TCTCTACCGCCACACCTCAAGT, 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: TTTACCGTCTCCATTAGCAACAA, 19-R: GTGAACAGAGGCGAATCAAACAT; 20-F: GAACCACCCAAGACCAAACT, 20-R: AGCGATGACATCAAACACCT; 21-F: ACATTATGTCCCATCATACGAGGTG, 21-R: TTGACTGTTAAGGAGAATCCCAGAG; 22-F: ATACAAGTTGCTGCGGATGATTC, 22-R: GAAGAGGAGGAAGGTGATGAAGG; 23-F: TCACAAAGTAAACATGCTTCTGCT, 23-R: TTTCTCTTCATTTGGTGAAGTGCT; 24-F: CTCAGGAATCACTTTCTTTGGT, 24-R: TCCAGTAGGTTTGTGTTTGCCTT; (3) taking the first round of PCR products as templates, performing a second round of PCR reaction by using a universal P7 primer and a universal P5 primer, and obtaining second round of PCR products; (4) mixing the second round of PCR products in equal amounts and then performing sequencing by using a high-throughput sequencer; (5) performing quality control on the sequencing data, performing genome alignment, SNPs identification and reads calculation on the sequencing data, judging the genotype of the hybrid individual, and then identifying the genetic composition of the offspring of the hybrid between the mandarin fish and the topmouth culter.

2. The method of claim 1, wherein, The first round of PCR system is as follows: 10 ng / μL DNA template 2 μL; 10 μM upstream primer pool 1 μL; 10 μM downstream primer pool 1 μL; 2×PCR Ready Mix 15 μL, the total volume is 25 μL; the first round of PCR reaction program is as follows: 98 ℃ pre-denaturation for 3 min; then performing 8 cycles, the condition is 98 ℃ denaturation for 30 s, 50 ℃ annealing for 30 s, 72 ℃ extension for 30 s; followed by performing 25 cycles, the condition is 98 ℃ denaturation for 30 s, 66 ℃ annealing for 30 s, 72 ℃ extension for 30 s; finally 72 ℃ extension for 5 min.

3. The method of claim 1, wherein, The second round of PCR system is as follows: 10 ng / μL DNA template 2 μL; 10 μM molecular tag-containing universal P7 primer 1 μL; 10 μM universal P5 primer 1 μL; 2×PCR Ready Mix 15 μL, total volume 30 μL; the second round of PCR reaction program is as follows: 98 ℃ pre-denaturation 5 min; then perform 5 cycles of program, the condition is denaturation 94 ℃ 30 s, 55 ℃ annealing 20 s, 72 ℃ extension 30 s; final 72 ℃ extension 5 min; after completion, 4 ℃ incubation.

4. The method of claim 1, wherein, After the first round or the second round of PCR reaction is completed, the PCR product is purified and recovered by using AMPure XP magnetic beads.

5. The method of claim 1, wherein, The PCR reaction is carried out on a GeneAmp PCR System 2700 thermal cycler.

6. The method of claim 1, wherein, The sequencing data is quality controlled by using fastq quality control software, and the sequencing data is genome aligned, SNP identified and read calculated by using bowtie2, samtools and bedtools software.

7. The method according to any one of claims 1 to 6, characterized in that, The process of judging the genotype of the hybrid individual is as follows: the specific genomic region of the mandarin fish and the topmouth culter is simultaneously used as a reference sequence, the base frequency detected by the DNA mixed sample of the mandarin fish and the topmouth culter F1, the mandarin fish and the topmouth culter at the same concentration is used as the basis for evaluating the error range of the detection result, and after filtering out the sites with insufficient credibility, all data results are classified and counted.

8. A pool of specific primers, characterized in that, The specific primer pool comprises 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: GGACCTATTCCTTTACCGACCA, 4-R: GCTGGCTAAAGGCTTTATCTGT; 5-F: GTGGTATGTCACCTTCCTTTTG, 5-R: CACGTCTACTATCCTCTTTGGG; 6-F: GGAACAAATACCTGAGCCTGAT, 6-R: CAGAATCTTTAACACCCGTGAG; 7-F: GAGTTCAGGGACAACAAAGATGT, 7-R: TCTCTGAGAAGACCATTAGCACC; 8-F: CTTCAACAGAATTGGAGGCATG, 8-R: GAGGGATGAAATCTTCCTTGGC; 9-F: GCTCACAGATGACCACAAACGT, 9-R: TGGCTCCAGAACAGAGAATGAA; 10-F: GAACCCCTCGTGCATCCAAC, 10-R: CACCGCATTCCCTCCCATCT; 11-F: TCTCTACCGCCACACCTCAAGT, 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: TTTACCGTCTCCATTAGCAACAA, 19-R: GTGAACAGAGGCGAATCAAACAT; 20-F: GAACCACCCAAGACCAAACT, 20-R: AGCGATGACATCAAACACCT; 21-F: ACATTATGTCCCATCATACGAGGTG, 21-R: TTGACTGTTAAGGAGAATCCCAGAG; 22-F: ATACAAGTTGCTGCGGATGATTC, 22-R: GAAGAGGAGGAAGGTGATGAAGG; 23-F: TCACAAAGTAAACATGCTTCTGCT, 23-R: TTTCTCTTCATTTGGTGAAGTGCT; 24-F: CTCAGGAATCACTTTCTTTGGT, 24-R: TCCAGTAGGTTTGTGTTTGCCTT.

9. Use of the specific primer pool of claim 8 for identifying the genetic composition of the offspring of a hybrid between Misgurnus fossilis and Ctenopharyngodon idellus. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

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