Primer pair for identifying oryzias fish and identification method

Abstract

The invention discloses a primer pair for identifying oryzias fish and an identification method, and belongs to the field of fish identification. The invention provides a pair of primer pairs, the sequences of the primer pairs are shown as SEQ ID NO: 1 and SEQ ID NO: 2, the primers are used for performing PCR amplification on genome DNA of a sample to be detected, the PCR amplification result is sequenced, a phylogenetic tree can be constructed according to the sequencing result to classify oryzias fishes, when the sequencing result and any sequence of SEQ ID NO: 3-SEQ ID NO: 23 are gathered into one oryzias sinensis, the oryzias sinensis is the oryzias sinensis, and when the sequencing result and any sequence of SEQ ID NO: 3-SEQ ID NO: 23 are gathered into one oryzias sinensis, the oryzias sinensis is the oryzias sinensis. The oryzias curvinotus is obtained when the sequencing result and any one sequence of SEQ ID NO: 24 to SEQ ID NO: 41 are gathered into one oryzias curvinotus, and the oryzias peistilatus is obtained when the sequencing result and any one sequence of SEQ ID NO: 42 to SEQ ID NO: 43 are gathered into one oryzias peistilatus, the oryzias peistilatus can be simply and efficiently classified and identified, and the identification accuracy is relatively high.

Description

Technical Field

[0001] The invention relates to a primer pair and an identification method for identifying fish of the genus Medaka, and belongs to the field of fish identification. Background Art

[0002] Medaka is a group of small fish belonging to the family Adrianichthyidae, order Cyprinodontiformes, and genus Oryzias. They are widely distributed in freshwater and coastal areas of continental and island areas in the western Pacific region. So far, 33 species have been described.

[0003] The main medaka species in the Pearl River Basin and adjacent areas of China are: Chinese medaka (Oryzias sinensis), fin-spotted medaka (Oryzias pectoralis) and bow-backed medaka (Oryzias curvinotus). Currently, the three fish species are mainly distinguished by their morphological characteristics. This method requires measurement of various characteristics of the fish, such as total body length, head length, head length / body height, body length / body height, post-anal length, mouth width and egg long diameter, etc. Not only is the measurement process complicated, but the data analysis process is also very cumbersome, and the morphological characteristics of each fish may change due to changes in the environment, thus causing the problem of inaccurate distinction. The present invention starts with molecular biology and provides a pair of primers. By using the primers for PCR and sequencing, Chinese medaka, fin-spotted medaka and bow-backed medaka can be accurately distinguished based on sequencing results. Summary of the invention

[0004] One of the purposes of the present invention is to provide a primer for identifying fish of the genus Medaka, wherein the upstream primer sequence of the primer pair is shown as SEQ ID NO:1, and the downstream primer sequence is shown as SEQ ID NO:2.

[0005] Another object of the present invention is to provide a method for identifying fish of the genus Medaka, which specifically comprises the following steps:

[0006] (1) Extract genomic DNA from the sample to be tested.

[0007] (2) Using the genomic DNA obtained in step (1) as a template, PCR amplification is performed using the primer pair shown in SEQ ID NO: 1 and SEQ ID NO: 2 to obtain a PCR product.

[0008] (3) Sequencing the PCR product obtained in step (2) and analyzing the sequencing results. When the sequencing results are clustered with any one of SEQ ID NO:3 to SEQ ID NO:23, it is Oryzias sinensis; when the sequencing results are clustered with any one of SEQ ID NO:24 to SEQ ID NO:41, it is Oryzias curvinotus; when the sequencing results are clustered with any one of SEQ ID NO:42 to SEQ ID NO:43, it is Oryzias pectoralis.

[0009] Beneficial Effects of the Invention

[0010] (1) The present invention can identify fish of the genus Medaka by performing one PCR and sequencing using a pair of primers. The method is simple, efficient and has a high accuracy rate. DETAILED DESCRIPTION

[0011] The technical solution of the present invention is further described below by specific implementation methods. However, the following embodiments are only simple examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0012] Example 1

[0013] 96 known species of medaka were mixed and classified and identified. The specific identification method is as follows:

[0014] (1) Genomic DNA of 96 fish was extracted using a DNA extraction kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., catalog number: DP304-02);

[0015] (2) Using the DNA obtained in step (1) as a template, PCR was performed using 2×Power Taq PCR MasterMix (purchased from Background Biotech Co., Ltd.), the PCR system is shown in Table 1, and the PCR program is shown in Table 2 to obtain a PCR product.

[0016] Table 1 PCR system

[0017] name Dosage Upstream primer 4μL Downstream primer 4μL 2×Power Taq PCR MasterMix 25μL DNA template 2μL <![CDATA[ddH 2 The]]> Up to 50 μL

[0018] Table 2 PCR program

[0019]

[0020] (2) Take 3 μL of the PCR product obtained in step (2) and perform electrophoresis on a 0.8% agarose gel, and select samples with specific bright bands for sequencing.

[0021] (4) The sequencing results were analyzed, and 21 haplotypes (SEQ ID NO:3-SEQ ID NO:23) were obtained from 45 Chinese medaka, 18 haplotypes (SEQ ID NO:24-SEQ ID NO:41) were obtained from 31 bow-backed medaka, and 2 haplotypes (SEQ ID NO:42-SEQ ID NO:43) were obtained from 20 medaka. Cluster analysis of the obtained haplotype sequences showed that the haplotype sequences from different medaka species were clustered into one branch, proving that the primers described in the present invention can accurately distinguish Chinese medaka, bow-backed medaka and medaka.

Claims

1. A primer pair for identifying fish of the genus Medaka, characterized in that: The upstream primer sequence of the primer pair is shown as SEQ ID NO: 1, and the downstream primer sequence is shown as SEQ ID NO:

2.

2. A method for identifying a fish of the genus Medaka, characterized in that: The specific steps include: (1) extracting genomic DNA from the sample to be tested; (2) using the genomic DNA obtained in step (1) as a template, performing PCR amplification using the primer pair shown in SEQ ID NO: 1 and SEQ ID NO: 2 to obtain a PCR product; (3) Sequencing the PCR product obtained in step (2) and analyzing the sequencing results. When the sequencing results are clustered with any one of SEQ ID NO:3 to SEQ ID NO:23, it is a Chinese medaka. When the sequencing results are clustered with any one of SEQ ID NO:24 to SEQ ID NO:41, it is a bow-backed medaka. When the sequencing results are clustered with any one of SEQ ID NO:42 to SEQ ID NO:43, it is a fin-spotted medaka.

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