Molecular marker for identifying subspecies of northern sea track of sika deer, identification method and application

A technology of molecular markers and sika deer, which is applied in biochemical equipment and methods, measurement/inspection of microorganisms, DNA/RNA fragments, etc., can solve the problems that there is no molecular marker identification method for sika deer subspecies, and achieve low cost and low cost. Cost and time saving effects

Pending Publication Date: 2021-01-12
INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are no molecular markers and

Method used

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  • Molecular marker for identifying subspecies of northern sea track of sika deer, identification method and application
  • Molecular marker for identifying subspecies of northern sea track of sika deer, identification method and application
  • Molecular marker for identifying subspecies of northern sea track of sika deer, identification method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1. Acquisition of specific SNP markers for identifying the Hokkaido subspecies of sika deer

[0024] 1. Screening of specific SNP loci of sika deer Hokkaido subspecies

[0025] Combining the mtDNA sequences of different subspecies samples of sika deer, such as the Northeast subspecies, Sichuan subspecies, South China subspecies, Taiwan subspecies, Hokkaido subspecies, Honshu subspecies, nominated subspecies and Yakushima subspecies, were compared by Mega6.0. For the analysis, the screening is mainly aimed at the loci shared by the same subspecies and specific to other subspecies, and 4 differential SNP loci are screened, such as figure 1 shown. The ND1 gene fragment, ATP6 gene fragment, ND4 gene fragment and ND6 gene fragment were intercepted with Mega6.0, and the sequence length was about 500bp, which was used to design identification primers.

[0026] 2. Primer design

[0027] Based on the intercepted target gene fragments, primers for identification were ...

Embodiment 2

[0041] Embodiment 2, the establishment of the method for identifying sika deer Hokkaido subspecies

[0042] According to the primer pair shown in the above SEQ ID NO.1-SEQ ID NO.8, PCR experiments were carried out to establish a method for identifying the Hokkaido subspecies and other subspecies of sika deer. The PCR amplification conditions are shown in Table 2.

[0043] Table 2 PCR amplification conditions

[0044]

[0045] The PCR reaction system is shown in Table 3.

[0046] Table 3 PCR system

[0047]

[0048] The PCR amplification process is: pre-denaturation at 94°C for 5 minutes; 30 cycles of denaturation at 94°C for 30 sec, annealing at 59°C for 30 sec, and extension at 72°C for 30 sec; extension at 72°C for 5 min, and storage at 4°C.

[0049] PCR results such as figure 2 , the PCR results of primer pair 1, primer pair 2, primer pair 3 and primer pair 4 have single and bright electrophoresis bands, and the experimental results show that the present invention...

Embodiment 3

[0050] Embodiment 3. The specific application of the method for identifying the Hokkaido subspecies of sika deer and other subspecies of the present invention

[0051] A total of 100 samples were selected from China and Japan, and genomic DNA was extracted respectively. Experimenter A randomly mixed these samples and renumbered them. Experimenter B used the primers designed by the present invention to carry out PCR amplification on these samples, and the amplified product Sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing. View the sequencing peak map through Bioedit7.0, the peak and valley of the sequencing result map are separated, there is no overlap, and there is no misreading, indicating that the sequencing result is reliable. The sequences were aligned using Mega6.0, and the bases of the specific SNP sites of the Hokkaido subspecies of sika deer were used as the basis for determination.

[0052] It is judged according to the mtDNA-specific SNP molecular m...

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Abstract

The invention provides a molecular marker for identifying subspecies of northern sea track of sika deer, an identification method and application, and aims at accurately identifying the subspecies ofthe northern sea track of the sika deer. Four SNP sites S-1 to S-4 of the molecular marker are respectively shown in the subspecies of the northern sea track of the sika deer, and are respectively shown in non-subspecies of the northern sea track of the sika deer. The invention also provides a method for identifying the subspecies of the northern sea track of the sika deer. The method comprises the following steps of: performing PCR amplification and sequencing on any one or more pairs of primer pairs 1 to 4, and identifying whether a sample to be tested is the subspecies of the northern sea track of the sika deer according to SNP marker site bases. The four SNP sites are positioned on four gene segments, and have the characteristics of high specificity and high stability. The identification detection accuracy is 100 percent. The invention provides a stable and reliable molecular detection method for identifying subspecies of sika deer resources. The molecular detection method has important theoretical and application values in the aspects of DNA fingerprint drawing of the sika deer, wild subspecies protection management and the like.

Description

technical field [0001] The invention relates to a molecular marker for identifying the Hokkaido subspecies of sika deer, an identification method and an application, and belongs to the technical field of molecular biology detection. Background technique [0002] The sika deer (Cervus nippon) belongs to Mammalia, Artiodactyla, Cervidae, and Cervus. It is a symbolic animal in the East Asian monsoon region and is distributed from the Ussuri River to Vietnam. Due to the destruction of the ecological environment and the shrinking habitat, the number of wild sika deer in my country is very rare. It is currently a Class I protected animal in my country and has also been included in the Red List of Endangered Species by the International Union for Conservation of Nature (IUCN). At present, the subspecies division of sika deer is mainly based on geographical distribution and morphological characteristics. There is great controversy and confusion in classification. There are 6 species...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/156
Inventor 邢秀梅董依萌王天骄李洋刘华淼
Owner INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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