Goose mitochondrial genome complete sequence acquisition method
By designing specific PCR primers and high-throughput sequencing methods, the process of obtaining the goose mitochondrial genome has been simplified, the accuracy has been improved and the cost has been reduced. It is suitable for ordinary laboratories and realizes rapid and accurate goose mitochondrial genome detection.
Patent Information
- Application Number
- CN202510491410.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-18
- Publication Date
- 2025-09-23
AI Technical Summary
The existing technology for obtaining the goose mitochondrial genome is cumbersome, inaccurate and costly, making it difficult to promote and apply in ordinary laboratories.
Specific PCR primers and high-throughput sequencing methods were designed. Goose mitochondrial DNA fragments were amplified using two pairs of primers, and high-throughput sequencing and bioinformatics splicing were performed to obtain the complete sequence of the goose mitochondrial genome.
The method simplifies the operation process, improves accuracy, reduces costs, is suitable for ordinary laboratories, and can quickly and accurately obtain goose mitochondrial genomes and detect mutation types and haplotypes.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for obtaining the full sequence of a goose mitochondrial genome. Background Art
[0002] As an important component of germplasm resources and even biodiversity, the protection and utilization of livestock and poultry genetic resources are of great significance for maintaining ecological balance and promoting sustainable agricultural development. Therefore, it is of great significance to analyze and evaluate the genetic diversity of local goose breeds.
[0003] Animal cells contain two genomic systems: the nuclear genome and the mitochondrial genome. Mitochondrial DNA has an independent genetic coding system that does not follow Mendel's laws of inheritance. Its genomic structure is simple, lacks homologous recombination, undergoes rapid sequence evolution, and is strictly maternally inherited. It is widely used in studies of animal origins and evolution, population genetic structure, species identification, and economic traits. Only a small sample of individuals is needed to reflect the genetic structure of a population. Goose mitochondria contain a D-loop region, 13 protein-coding genes, two rRNA genes (12S rRNA and 16S rRNA), and 22 tRNA genes.
[0004] The traditional method for obtaining the goose mitochondrial genome combines a primer-walking strategy, designing multiple pairs of overlapping primers for PCR amplification and Sanger sequencing. Finally, the sequenced fragments are spliced together to obtain the complete mitochondrial genome. The goose mitochondrial genome is 16,738 base pairs, and the effective read length of Sanger sequencing is typically around 700 base pairs. Including overlapping regions, more than 30 sequencing reactions are required to complete the sequence. This cumbersome process can also result in poor sequencing accuracy in some regions with high GC content.
[0005] With the development of second-generation sequencing technology and the rapid decline in sequencing costs, more and more researchers are using it to obtain goose mitochondrial genomes. This method involves randomly fragmenting the total sample DNA into small DNA fragments. These fragments are then connected to sequencing adapters at both ends and sequenced. Finally, the sequencing data is spliced together using bioinformatics methods to obtain sequence information for the entire mitochondrial genome. Because nuclear genomic DNA constitutes the vast majority of the total sample DNA, the mitochondrial genome accounts for a relatively small proportion of the second-generation sequencing data. To address the issue of insufficient sequencing data, the only option is to increase the total sequencing data volume, which leads to increased costs. Furthermore, the data processing and splicing process is cumbersome, requiring specialized bioinformatics expertise and extensive computing resources, making it difficult to apply in general laboratories.
[0006] Therefore, there is an urgent need to develop a method for obtaining the goose mitochondrial genome with simple procedures, high accuracy and low cost. Summary of the Invention
[0007] The technical problem that the present invention aims to solve is to address the deficiencies of the above-mentioned prior art. The present invention provides a method for obtaining the full sequence of the goose mitochondrial genome, comprising: (1) sample collection; (2) PCR amplification; (3) high-throughput sequencing; and (4) mitochondrial genome assembly. The method is easy to operate and has a high accuracy rate.
[0008] The present invention achieves the purpose of the present invention through the following technical solutions:
[0009] One of the purposes of the present invention is to provide a PCR primer for obtaining the complete sequence of the goose mitochondrial genome, the nucleotide sequence of the primer is:
[0010] The first pair of forward primers: 5'-CTGATCCGTACTAATTACCGCCA-3' (SEQ ID NO. 1);
[0011] The first pair of reverse primers: 5'-GGATTTTGTCGCAGTCTGATACG-3' (SEQ ID NO. 2);
[0012] The second pair of forward primers: 5'-CCCTAGTCCACCTAACCTTCCTA-3' (SEQ ID NO. 3);
[0013] The second pair of reverse primers: 5'-GACTACGTGTGAGATGATTCCGA-3' (SEQ ID NO. 4).
[0014] A second object of the present invention is to provide an application in a method for obtaining the full sequence of a goose mitochondrial genome, comprising: (1) extracting genomic DNA from a goose to be tested; (2) performing PCR amplification using the primers described in the present invention to obtain two goose mitochondrial DNA fragments; (3) performing high-throughput sequencing; and (4) assembling the mitochondrial genome to obtain the full-length mitochondrial DNA.
[0015] In the method described in the present invention, step (1) extracting the genomic DNA of the goose to be tested can be performed according to conventional methods in the art. For example, the genomic DNA of the goose to be tested can be extracted from materials including but not limited to feathers, blood, tissues, organs, etc.
[0016] In the method of the present invention, the PCR reaction system in step (2) can be a conventional system in the art. In a specific embodiment of the present invention, the PCR reaction system can be, for example, 25 μL of 2×Vazyme LAmp Master Mix (Nanjing Novozyme Biotechnology Co., Ltd.), 2 μL each of 10 μmol / L forward and reverse primers, 2 μL of 50-100 μg / ml template DNA, and 19 μL of ultrapure water.
[0017] In the method described in the present invention, the PCR reaction procedure in step (2) can be a conventional system in the art. In a specific embodiment of the present invention, it is: 95°C for 5 min, 35 cycles of (95°C for 30 s, 66°C for 9 min), and 72°C for 10 min.
[0018] The method of the present invention, step (3) Chinese library construction can be a conventional operation in the art. In a specific embodiment of the present invention,
[0019] (1) Preparation of enzyme digestion system
[0020] a) Prepare sterile, enzyme-free PCR tubes and set up the enzyme digestion reaction system on ice; input 50 ng of amplified product, 1 μL of Tn5 Enzyme, and add Working Buffer to 20 μL;
[0021] b) After the enzyme digestion system is prepared, place the PCR tube on a PCR instrument with a pre-heated cover at 70°C and a temperature of 55°C, and perform the enzyme digestion reaction for 7 minutes.
[0022] (2) Termination of enzyme digestion reaction
[0023] a) After the enzyme digestion reaction is complete, place the tube on ice immediately and prepare the enzyme digestion stop reaction system: 20 μL of the enzyme digestion system from the previous step and 1 μL of Stop Buffer.
[0024] b) Place the PCR tube on a PCR instrument pre-heated at 70°C and 55°C, and perform the end-enzyme digestion program for 5 minutes.
[0025] (3) Purification and recovery of enzyme digestion products
[0026] a) Preparation: Remove DNA Selection Beads magnetic beads from the refrigerator and equilibrate them at room temperature for at least 30 minutes. Meanwhile, prepare 80% ethanol.
[0027] b) Vortex or invert the beads thoroughly to ensure thorough mixing.
[0028] c) Pipette 20 μl of DNA Selection Beads (0.9X, Beads:DNA=0.9:1) into the product after enzyme digestion, vortex or pipette to mix, and incubate at room temperature for 5 minutes.
[0029] d) Briefly centrifuge the PCR tube and place it on a magnetic rack to separate the beads and liquid. After the solution has clarified (approximately 5 minutes), carefully remove the supernatant.
[0030] e) Keeping the PCR tube in the magnetic rack, add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads. Incubate at room temperature for 30 seconds, then carefully remove the supernatant.
[0031] f) Repeat step e and rinse once more.
[0032] g) Keep the PCR tube in the magnetic rack at all times, open the lid and air-dry the magnetic beads until they just begin to crack (no more than 5 minutes).
[0033] h) Remove the PCR tube from the magnetic rack and add 15 μl of ddH2O. Vortex or gently pipette to mix thoroughly. Let stand at room temperature for 5 minutes. Briefly centrifuge the PCR tube and place it on the magnetic rack. Once the solution has cleared (approximately 3 minutes), carefully transfer 15 μl of the supernatant to a new PCR tube for the next amplification step.
[0034] (4) Library amplification
[0035] a) Prepare the amplification system: 10 μL of purified product, 15 μL of 2X High-Fidelity Mix, 2 μL of UDI, and add HO to 30 μL.
[0036] b) Amplification program: 72°C for 3 min, 98°C for 3 min, 7-15 cycles of (98°C for 30 s, 60°C for 30 s, 72°C for 30 s), 72°C for 3 min.
[0037] (5) Library quality control
[0038] Typically, the quality of the constructed library can be evaluated by concentration detection and length distribution detection.
[0039] (6) High-throughput sequencing
[0040] The constructed NGS library was sequenced using an Illumina MiSeq sequencer in SE150 or PE150 mode.
[0041] In the method of the present invention, the mitochondrial genome assembly in step (4) can be a conventional operation in the art. In a specific embodiment of the present invention,
[0042] (1) Use cutadapt (v 1.2.1) software to remove any partial sequence containing sequencing adapter sequences;
[0043] (2) PRINSEQ-lite (v 0.20.3) software was used to perform quality control on the remaining sequences. Bases with a quality threshold lower than 20 were deleted from the 3' end to the 5' end of the sequence. The remaining sequences were considered to have passed the quality control.
[0044] (3) SPAdes (v3.15.0) software was used to assemble the sequences that passed quality control, and the default parameters were selected to output the spliced sequences.
[0045] The present invention provides the application of the PCR primers or the method for obtaining the full sequence of the goose mitochondrial genome in the present invention in detecting different mutation types or haplotypes.
[0046] Compared with the prior art, the present invention has the following advantages:
[0047] (1) Conventional sequencing requires the design of more than 30 amplification or sequencing reactions to complete the test. The present invention only requires two pairs of primers to cover the entire length of the goose mitochondrial genome through the designed PCR primers, which greatly reduces the workload.
[0048] (2) Directly construct a library of two long-fragment PCR products for high-throughput sequencing, which has high accuracy and is priced at only one-fifth of conventional sequencing, one-tenth of the cost of second-generation sequencing, and one-twenty-fifth of the cost of third-generation sequencing. The specific comparison is shown in Table 1.
[0049] Table 1 Comparison of different methods for obtaining full-length mitochondrial genomes
[0050]
[0051] (3) The primers of the present invention have little restriction on goose breeds, so there is no restriction on the specific breed of the geese described in the present invention.
[0052] (4) The method for obtaining the goose mitochondrial genome of the present invention obtains two fragments of the full-length mitochondrial DNA by one-step PCR amplification from the total DNA, which can ensure the accurate amplification of the goose mitochondrial genome and prevent it from being contaminated by homologous sequences in the goose genome, making mutation detection more sensitive and accurate and saving time.
[0053] (5) The mitochondrial DNA library constructed by the present invention is subjected to high-throughput sequencing and bioinformatics analysis to obtain the mitochondrial genome DNA sequence of the test sample, thereby detecting different mutation types and haplotypes. BRIEF DESCRIPTION OF THE DRAWINGS
[0054] Figure 1The structure of the assembled goose mitochondrial genome.
[0055] Figure 2 Phylogenetic relationships of mitochondrial genomes of local goose breeds DETAILED DESCRIPTION
[0056] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.
[0057] Example 1
[0058] 1 Establishment of goose mitochondrial genome method
[0059] 1.1 Primer design
[0060] With reference to the standard sequence of goose mitochondrial genome (NCBI database ID number: NC011196), two pairs of primers were designed to cover the full length of goose mitochondrial genome. The length of amplified product of the first pair was 8363 bp, and the length of amplified product of the second pair was 8678 bp, with an overlapping region of 298 bp.
[0061] The first pair of forward primers: 5′-CTGATCCGTACTAATTACCGCCA-3′;
[0062] The first pair of reverse primers: 5′-GGATTTTGTCGCAGTCTGATACG-3′;
[0063] The second pair of forward primers: 5′-CCCTAGTCCACCTAACCTTCCTA-3′;
[0064] The second pair of reverse primers: 5'-GACTACGTGTGAGATGATTCCGA-3'.
[0065] 1.2 PCR amplification
[0066] The primers designed by the present invention were used to amplify the Sichuan white goose DNA preserved by the research group.
[0067] The PCR reaction system is:
[0068] 25 μL of 2×Vazyme LAmp Master Mix (Nanjing Novozyme Biotechnology Co., Ltd.), 2 μL of 10 μmol / L forward and reverse primers, 2 μL of 50-100 μg / ml template DNA, and 19 μL of ultrapure water.
[0069] The PCR reaction program was: 95°C for 5 min, 35 cycles of (95°C for 30 s, 66°C for 9 min), and 72°C for 10 min.
[0070] 1.3 High-throughput sequencing
[0071] (1) Preparation of enzyme digestion system
[0072] a) Prepare sterile, enzyme-free PCR tubes and prepare the enzyme digestion reaction system on ice. Input 50 ng of amplified product, 1 μL of Tn5 Enzyme, and add 20 μL of Working Buffer.
[0073] b) After the enzyme digestion system is prepared, place the PCR tube on a PCR instrument with a pre-heated cover at 70°C and a temperature of 55°C, and perform the enzyme digestion reaction for 7 minutes.
[0074] (2) Termination of enzyme digestion reaction
[0075] a) After the enzyme digestion reaction is complete, place the tube on ice immediately and prepare the enzyme digestion stop reaction system: 20 μL of the enzyme digestion system from the previous step and 1 μL of Stop Buffer.
[0076] b) Place the PCR tube on a PCR instrument pre-heated at 70°C and 55°C, and perform the end-enzyme digestion program for 5 minutes.
[0077] (3) Purification and recovery of enzyme digestion products
[0078] a) Preparation: Remove the DNA Selection Beads magnetic beads from the refrigerator and equilibrate them at room temperature for at least 30 minutes. Meanwhile, prepare 80% ethanol.
[0079] b) Vortex or invert the beads thoroughly to ensure thorough mixing.
[0080] c) Pipette 20 μl of DNA Selection Beads (0.9X, Beads:DNA=0.9:1) into the product after enzyme termination, vortex or pipette to mix, and incubate at room temperature for 5 minutes.
[0081] d) Briefly centrifuge the PCR tube and place it on a magnetic rack to separate the beads and liquid. After the solution has clarified (approximately 5 minutes), carefully remove the supernatant.
[0082] e) Keeping the PCR tube in the magnetic rack, add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads. Incubate at room temperature for 30 seconds, then carefully remove the supernatant.
[0083] f) Repeat step e and rinse once more.
[0084] g) Keep the PCR tube in the magnetic rack at all times, open the lid and air-dry the magnetic beads until they just begin to crack (no more than 5 minutes).
[0085] h) Remove the PCR tube from the magnetic rack and add 15 μl of ddH2O. Vortex or gently pipette to mix thoroughly. Let stand at room temperature for 5 minutes. Briefly centrifuge the PCR tube and place it on the magnetic rack. Once the solution has cleared (approximately 3 minutes), carefully transfer 15 μl of the supernatant to a new PCR tube for the next amplification step.
[0086] (4) Library amplification
[0087] a) Prepare the amplification system: 10 μL of purified product, 15 μL of 2X High-Fidelity Mix, 2 μL of UDI, and add HO to 30 μL.
[0088] b) Amplification program: 72°C for 3 min, 98°C for 3 min, 7-15 cycles of (98°C for 30 s, 60°C for 30 s, 72°C for 30 s), 72°C for 3 min.
[0089] (5) Library quality control
[0090] Typically, the quality of the constructed library can be evaluated by concentration detection and length distribution detection.
[0091] (6) High-throughput sequencing
[0092] The constructed NGS library was sequenced using an Illumina MiSeq sequencer in SE150 or PE150 mode.
[0093] 1.4 Mitochondrial genome assembly
[0094] (1) Use cutadapt (v 1.2.1) software to remove any partial sequence containing sequencing adapter sequences;
[0095] (2) PRINSEQ-lite (v 0.20.3) software was used to perform quality control on the remaining sequences. Bases with a quality threshold lower than 20 were deleted from the 3' end to the 5' end of the sequence. The remaining sequences were considered to have passed the quality control.
[0096] (3) SPAdes (v3.15.0) software was used to assemble the sequences that passed the quality control. The default parameters were selected and the spliced sequences were output. The assembled Sichuan white goose mitochondrial genome was 16742 bp long. After gene annotation, it contained 22 tRNA genes, 2 rRNA genes (12S rRNA and 16S rRNA), 13 protein-coding genes and 1 D-loop region ( Figure 1 ).
[0097] 1.5 Comparison with third-generation sequencing results
[0098] The DNA samples of Sichuan white geese were directly subjected to third-generation sequencing, and the sequence identity with the present invention was 100%, indicating that the method of the present invention can ensure the accurate amplification of the goose mitochondrial genome and is not contaminated by homologous sequences of the goose nuclear genome.
[0099] Example 2 Phylogenetic Analysis of Local Goose Breeds
[0100] 2.1 Sample collection
[0101] Blood was collected from 11 local goose breeds, including Xupu goose, Wugang bronze goose, Wanxi white goose, Youjiang goose, Wugang bronze grey goose, Daozhou grey goose, Zhedong white goose, Sichuan white goose, Hepu goose, Mayang white goose and Yan goose, and their DNA was extracted.
[0102] 2.2 PCR amplification
[0103] The primers designed by the present invention are used to amplify the goose mitochondrial genome.
[0104] The first pair of forward primers: 5′-CTGATCCGTACTAATTACCGCCA-3′;
[0105] The first pair of reverse primers: 5′-GGATTTTGTCGCAGTCTGATACG-3′;
[0106] The second pair of forward primers: 5′-CCCTAGTCCACCTAACCTTCCTA-3′;
[0107] The second pair of reverse primers: 5'-GACTACGTGTGAGATGATTCCGA-3'.
[0108] The PCR reaction system is:
[0109] 25 μL of 2×Vazyme LAmp Master Mix (Nanjing Novozyme Biotechnology Co., Ltd.), 2 μL of 10 μmol / L forward and reverse primers, 2 μL of 50-100 μg / ml template DNA, and 19 μL of ultrapure water.
[0110] The PCR reaction program was: 95°C for 5 min, 35 cycles of (95°C for 30 s, 66°C for 9 min), and 72°C for 10 min.
[0111] 2.3 High-throughput sequencing
[0112] (1) Preparation of enzyme digestion system
[0113] a) Prepare sterile, enzyme-free PCR tubes and prepare the enzyme digestion reaction system on ice. Input 50 ng of amplified product, 1 μL of Tn5 Enzyme, and add 20 μL of Working Buffer.
[0114] b) After the enzyme digestion system is prepared, place the PCR tube on a PCR instrument with a pre-heated cover at 70°C and a temperature of 55°C, and perform the enzyme digestion reaction for 7 minutes.
[0115] (2) Termination of enzyme digestion reaction
[0116] a) After the enzyme digestion reaction is complete, place the tube on ice immediately and prepare the enzyme digestion stop reaction system: 20 μL of the enzyme digestion system from the previous step and 1 μL of Stop Buffer.
[0117] b) Place the PCR tube on a PCR instrument pre-heated at 70°C and 55°C, and perform the end-enzyme digestion program for 5 minutes.
[0118] (3) Purification and recovery of enzyme digestion products
[0119] a) Preparation: Remove the DNA Selection Beads magnetic beads from the refrigerator and equilibrate them at room temperature for at least 30 minutes. Meanwhile, prepare 80% ethanol.
[0120] b) Vortex or invert the beads thoroughly to ensure thorough mixing.
[0121] c) Pipette 20 μl of DNA Selection Beads (0.9X, Beads:DNA=0.9:1) into the product after enzyme termination, vortex or pipette to mix, and incubate at room temperature for 5 minutes.
[0122] d) Briefly centrifuge the PCR tube and place it on a magnetic rack to separate the beads and liquid. After the solution has clarified (approximately 5 minutes), carefully remove the supernatant.
[0123] e) Keeping the PCR tube in the magnetic rack, add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads. Incubate at room temperature for 30 seconds, then carefully remove the supernatant.
[0124] f) Repeat step e and rinse once more.
[0125] g) Keep the PCR tube in the magnetic rack at all times, open the lid and air-dry the magnetic beads until they just begin to crack (no more than 5 minutes).
[0126] h) Remove the PCR tube from the magnetic rack and add 15 μl of ddH2O. Vortex or gently pipette to mix thoroughly. Let stand at room temperature for 5 minutes. Briefly centrifuge the PCR tube and place it on the magnetic rack. Once the solution has cleared (approximately 3 minutes), carefully transfer 15 μl of the supernatant to a new PCR tube for the next amplification step.
[0127] (4) Library amplification
[0128] a) Prepare the amplification system: 10 μL of purified product, 15 μL of 2X High-Fidelity Mix, 2 μL of UDI, and add HO to 30 μL.
[0129] b) Amplification program: 72°C for 3 min, 98°C for 3 min, 7-15 cycles of (98°C for 30 s, 60°C for 30 s, 72°C for 30 s), 72°C for 3 min.
[0130] (5) Library quality control
[0131] Typically, the quality of the constructed library can be evaluated by concentration detection and length distribution detection.
[0132] (6) High-throughput sequencing
[0133] The constructed NGS library was sequenced using an Illumina MiSeq sequencer in SE150 or PE150 mode.
[0134] 2.4 Mitochondrial genome assembly
[0135] (1) Use cutadapt (v 1.2.1) software to remove any partial sequence containing sequencing adapter sequences;
[0136] (2) PRINSEQ-lite (v 0.20.3) software was used to perform quality control on the remaining sequences. Bases with a quality threshold lower than 20 were deleted from the 3' end to the 5' end of the sequence. The remaining sequences were considered to have passed the quality control.
[0137] (3) SPAdes (v3.15.0) software was used to assemble the sequences that passed quality control, and the default parameters were selected to output the spliced sequences.
[0138] 2.5 Phylogenetic analysis
[0139] After obtaining the mitochondrial genome sequence, MAFFT (v7.520) software was used for comparative analysis, and DnaSP (v6.12.03) software was used to count the number of variant sites and haplotypes. MEGA (v11.0.13) software was used to construct a phylogenetic tree using the neighbor-joining (NJ) method.
[0140] The mitochondrial genome lengths of the 11 local goose breeds ranged from 16,739 to 16,744 bp, with a total of 379 variant sites found, including 123 single mutation sites and 256 parsimony informative sites. A total of 11 haplotypes were identified, and phylogenetic analysis of the 11 haplotypes revealed that the Mayang White Goose and the Yan Goose clustered together, while the other breeds clustered together ( Figure 2 ).
[0141] The above results show that the present invention can quickly, accurately and efficiently obtain the goose mitochondrial genome, detect different mutation types and haplotypes, and is simple and easy to operate.
Claims
1. A method for obtaining the complete sequence of the goose mitochondrial genome using PCR primers, wherein the two pairs of primers are as follows: The first pair of forward primers: 5'-CTGATCCGTACTAATTACCGCCA-3' (SEQ ID NO. 1); The first pair of reverse primers: 5'-GGATTTTGTCGCAGTCTGATACG-3' (SEQ ID NO. 2); The second pair of forward primers: 5'-CCCTAGTCCACCTAACCTTCCTA-3' (SEQ ID NO. 3); The second pair of reverse primers: 5'-GACTACGTGTGAGATGATTCCGA-3' (SEQ ID NO. 4).
2. Use of the PCR primers according to claim 1 in obtaining goose mitochondrial genome.
3. A method for obtaining the complete sequence of goose mitochondrial genome, characterized in that: The method comprises the following steps: (1) extracting genomic DNA from the goose to be tested; (2) performing PCR amplification using primers to obtain two goose mitochondrial DNA fragments; (3) performing high-throughput sequencing; and (4) assembling the mitochondrial genome to obtain the full-length mitochondrial DNA. The primer sequences used in step (2) are as follows: The first pair of forward primers: 5'-CTGATCCGTACTAATTACCGCCA-3' (SEQ ID NO. 1); The first pair of reverse primers: 5'-GGATTTTGTCGCAGTCTGATACG-3' (SEQ ID NO. 2); The second pair of forward primers: 5'-CCCTAGTCCACCTAACCTTCCTA-3' (SEQ ID NO. 3); The second pair of reverse primers: 5'-GACTACGTGTGAGATGATTCCGA -3' (SEQ ID NO. 4).
4. The method for obtaining the complete goose mitochondrial genome sequence according to claim 3, characterized in that: Step (1) extracting genomic DNA from feathers, blood, tissues or organs of the goose to be tested.
5. The method for obtaining the complete sequence of goose mitochondrial genome according to claim 3, characterized in that: The PCR reaction system in step (2) is 25 μL of 2×Vazyme LAmp Master Mix, 2 μL each of 10 μmol / L forward and reverse primers, 2 μL of 50-100 μg / ml template DNA, and 19 μL of ultrapure water.
6. The method for obtaining the complete sequence of goose mitochondrial genome according to claim 3, characterized in that: The PCR reaction program in step 2) was: 95°C for 5 min, 35 cycles of (95°C for 30 s, 66°C for 9 min), and 72°C for 10 min.
7. The method for obtaining the complete sequence of goose mitochondrial genome according to claim 3, characterized in that: Step 3) Chinese library construction includes the following steps: (1) Preparation of enzyme digestion system a) Prepare the enzyme digestion reaction system: input 50 ng of amplified product, 1 μL of Tn5 Enzyme, and add working buffer to 20 μL. b) After the enzyme digestion system is prepared, place the PCR tube on a PCR instrument with a pre-heated cover at 70°C and 55°C, and perform the enzyme digestion reaction for 7 minutes; (2) Termination of enzyme digestion reaction a) After the enzyme digestion reaction is completed, place the tube on ice immediately and prepare the enzyme digestion termination reaction system: 20 μL of the enzyme digestion system from the previous step and 1 μL of Stop Buffer; b) Place the PCR tube in a PCR instrument pre-heated at 70°C and 55°C and perform the end-enzyme digestion program for 5 minutes; (3) Purification and recovery of enzyme digestion products a) Preparation: Remove DNA Selection Beads from the refrigerator and equilibrate at room temperature for at least 30 minutes. Meanwhile, prepare 80% ethanol. b) Vortex or invert the beads thoroughly to ensure thorough mixing; c) Pipette 20 μl of DNA Selection Beads (0.9X, Beads:DNA = 0.9:1) into the product after enzyme digestion, vortex or pipette to mix, and incubate at room temperature for 5 minutes; d) Briefly centrifuge the PCR tube and place it on a magnetic rack to separate the beads and liquid. Once the solution has clarified (approximately 5 minutes), carefully remove the supernatant. e) Keeping the PCR tube in the magnetic rack, add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads. Incubate at room temperature for 30 seconds, then carefully remove the supernatant. f) Repeat step e and rinse once more; g) Keep the PCR tube in the magnetic rack at all times and air-dry the magnetic beads with the lid open until they just begin to crack, no more than 5 minutes. h) Remove the PCR tube from the magnetic rack, add 15 μl of ddH2O, vortex or gently pipette to mix thoroughly, and let it stand at room temperature for 5 minutes. Briefly centrifuge the PCR tube and place it on the magnetic rack to let it stand. Once the solution has clarified, carefully transfer 15 μl of the supernatant to a new PCR tube for the next amplification step. (4) Library amplification a) Prepare the amplification system: 10 μL of purified product, 15 μL of 2X High-Fidelity Mix, 2 μL of UDI, and add HO to 30 μL. b) Amplification program: 72°C for 3 min, 98°C for 3 min, 7-15 cycles of (98°C for 30 s, 60°C for 30 s, 72°C for 30 s), 72°C for 3 min; (5) Library quality control The constructed library is quality evaluated by concentration detection and length distribution detection; (6) High-throughput sequencing The constructed NGS library was sequenced using an Illumina MiSeq sequencer in SE150 or PE150 mode.
8. The method for obtaining the complete goose mitochondrial genome sequence according to claim 3, characterized in that: The mitochondrial genome assembly in step 4) includes the following steps: (1) Use cutadapt software to remove any partial sequence containing sequencing adapter sequences; (2) Use PRINSEQ-lite software to perform quality control on the remaining sequences. Delete bases with a quality threshold lower than 20 from the 3' end to the 5' end of the sequence. The remaining sequences are considered to have passed the quality control. (3) Use SPAdes software to assemble the sequences that have passed quality control, select the default parameters, and output the spliced sequences.
9. Use of the PCR primers according to claim 1 or the method for obtaining the full sequence of the goose mitochondrial genome according to any one of claims 3 to 8 in detecting different mutation types of goose mitochondrial genes.
10. Use of the PCR primers according to claim 1 or the method for obtaining the complete goose mitochondrial genome sequence according to any one of claims 3 to 8 in haplotype detection.