Primers, kit and method for identifying gender of chinese soft-shelled turtle and application thereof

Abstract

The application relates to the technical field of Chinese soft-shelled turtles, in particular to primers, a kit and a method for identifying the genders of Chinese soft-shelled turtles and application. The genomic DNA of male and female individuals of Chinese soft-shelled turtles is respectively subjected to PCR amplification by using the primer pair, and male-specific fragments and female-specific fragments can be obtained. The primer pair, the kit and the method provided in the embodiments can effectively distinguish female individuals and male individuals of Chinese soft-shelled turtles.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of Chinese soft-shelled turtle, in particular to primers, kits, methods and applications for identifying the gender of Chinese soft-shelled turtle. BACKGROUND

[0002] Chinese soft-shelled turtle (Pelodiscus sinensis) is also known as turtle, round fish, and king turtle. It belongs to Reptilia, Testudines, Trionychidae, and Trionychia. Chinese soft-shelled turtle of Yellow River strain is mainly distributed in the Yellow River basin of China, has large body, wide skirt, and slightly yellow body color, and has good growth and reproduction. Chinese soft-shelled turtle of Japanese strain is a pure breed of Chinese soft-shelled turtle introduced from China to Japan in the early 1960s, which is bred through careful selection and purification. Subsequently, this breeding variety was introduced from Japan to China and became an excellent Chinese soft-shelled turtle strain adapted to China. Chinese soft-shelled turtle of Huaihe River strain is mainly distributed along the coast of the Huaihe River basin in Anhui, has a soil yellow or yellow-brown back, a wide and thick skirt, and a growth rate second only to Japanese strain and Yellow River strain. As an excellent local breed, it is increasingly valued. SUMMARY

[0003] Based on this, the inventors of the present application sequenced the genomes of female and male Chinese soft-shelled turtles and found that there is a female-specific fragment between female and male Chinese soft-shelled turtles. The genomic DNA of male Chinese soft-shelled turtles was amplified by PCR using primer pair (SDR-1-F / SDR-1-R), and the results showed that one band was amplified in male Chinese soft-shelled turtles, while two bands were amplified in female Chinese soft-shelled turtles. This indicates that the primer pair, kit and method provided in the embodiments can effectively distinguish female and male Chinese soft-shelled turtles.

[0004] Therefore, the embodiments of the present application disclose at least the following technical solutions:

[0005] In a first aspect, the embodiments disclose a primer pair, which comprises a DNA as shown in SEQ ID NO: 1 and a DNA as shown in SEQ ID NO: 2. The genomic DNA of Chinese soft-shelled turtle is amplified by PCR using the primer pair, and the gender of Chinese soft-shelled turtle can be identified according to the amplification product.

[0006] In a second aspect, the embodiments disclose a kit. The kit comprises the primer pair of the second aspect and other reagents for PCR amplification.

[0007] In a third aspect, the embodiments disclose a method for identifying the gender of Chinese soft-shelled turtle. The method comprises performing PCR amplification with the genomic DNA of Chinese soft-shelled turtle as a template, and identifying the gender of Chinese soft-shelled turtle according to the amplification product.

[0008] In a fourth aspect, the embodiments disclose the application of the above primer pair, the above kit or the above method in the gender identification of Chinese soft-shelled turtle and / or breeding using gender identification. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 Electrophoresis result of the method for identifying the gender of Pelodiscus sinensis provided in the examples. In the figure, M is a marker, lanes 1-8 are results of male individuals of Pelodiscus sinensis, and lanes 9-16 are results of female individuals of Pelodiscus sinensis.

[0010] Figure 2 Sequencing peak chart of the amplified product that is common to both male and female individuals of Pelodiscus sinensis identified by the method for identifying the gender of Pelodiscus sinensis provided in the examples.

[0011] Figure 3 Sequencing peak chart of the amplified product that is unique to female individuals of Pelodiscus sinensis identified by the method for identifying the gender of Pelodiscus sinensis provided in the examples. DETAILED DESCRIPTION

[0012] In order to make the purposes, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with examples. It should be understood that the specific examples described herein are only used to explain the present application and not to limit the present application. The reagents not specifically described in the present application are conventional reagents and can be obtained from commercial channels; the methods not specifically described are conventional experimental methods and can be known from the prior art.

[0013] In the following experimental steps, unless otherwise specified, all operations are performed according to the methods provided in the Guide to Molecular Cloning (3rd Edition, Huang Peitang et al., Beijing: Science Press, 2002).

[0014] The inventors of the present application sequenced the genomes of female and male individuals of Pelodiscus sinensis and found that there is a female-specific fragment between female and male individuals of Pelodiscus sinensis. The genomic DNA of male individuals of Pelodiscus sinensis is subjected to PCR amplification using a primer pair (SDR-1-F / SDR-1-R), and a male-female differential fragment can be obtained. This indicates that the primer pair, kit and method provided in the examples can effectively distinguish female and male individuals of Pelodiscus sinensis.

[0015] The examples disclose that the primer pair comprises a DNA as shown in SEQ ID NO: 1 and a DNA as shown in SEQ ID NO: 2. The genomic DNA of Pelodiscus sinensis is subjected to PCR amplification using the primer pair, and the gender of Pelodiscus sinensis can be identified according to the amplified product.

[0016] The examples disclose a kit comprising the above-mentioned primer pair and other PCR amplification reagents. In some examples, the other PCR amplification reagents comprise 2x Phanta Flash Master Mix (Vazyme).

[0017] The embodiments disclose a method for identifying the gender of Chinese softshell turtles. The method comprises PCR amplification using genomic DNA of a Chinese softshell turtle to be tested as a template and identifying the gender of the Chinese softshell turtle to be tested according to the amplification product.

[0018] In some embodiments, the step of identifying the gender of the Chinese softshell turtle to be tested according to the amplification product comprises: performing electrophoresis detection on the amplification product, if one band is detected, the Chinese softshell turtle to be tested is a male individual; and if at least two bands are detected, the Chinese softshell turtle to be tested is a female individual.

[0019] In some embodiments, the step of identifying the gender of the Chinese softshell turtle to be tested according to the amplification product comprises:

[0020] performing sequencing on the amplification product, if only the sequence shown in SEQ ID NO: 3 is detected, the Chinese softshell turtle to be tested is a male individual; and if the sequences shown in SEQ ID NO: 3 and 4 are detected, the Chinese softshell turtle to be tested is a female individual.

[0021] The embodiments disclose application of the primer pair, the kit or the method to gender identification of Chinese softshell turtles and / or breeding by gender identification.

[0022] The present application will be further described in conjunction with more specific embodiments. Of course, the following embodiments should not be construed as limiting the present application.

[0023] Genomic DNA of muscle tissue of a Chinese softshell turtle individual is collected, and a primer pair shown in SEQ ID NO: 4 and 5 is used for PCR amplification. The PCR reaction system is 25 μL, comprising: 1 μL (100 ng / μL) of genomic DNA, 12.5 μL of 2x PhantaFlash Master Mix (Vazyme, Catalog #: P510), 1 μL of an upstream primer, 1 μL of a downstream primer and 9.5 μL of ddH2O. The PCR reaction program is: 98 ℃ for 30 s, 32 cycles of 98 ℃ for 10 s, 58 ℃ for 5 s, 72 ℃ for 5 s, and 72 ℃ for 1 min. The amplification product is detected by 1.2% agarose gel electrophoresis, and the gender of the Chinese softshell turtle to be tested is identified according to the band of the amplification product in the electrophoresis detection.

[0024] The steps for extracting genomic DNA include: taking approximately 0.5g of muscle tissue, thoroughly mincing it, and placing it in a 1.5mL centrifuge tube. Adding 500μL Hombuffer buffer and 10μL proteinase K, digesting at 55℃ for at least 3 hours, inverting and shaking once every 1 hour. Adding 500μL NaCl (4.5M) and 300μL chloroform, inverting and mixing for 20 minutes. Centrifuging at 13000rpm for 10 minutes. After centrifugation, aspirating 500μL of the supernatant and transferring it to a new tube, avoiding the middle layer. Adding 350μL isopropanol, gently inverting and mixing until a flocculent precipitate forms, allowing it to stand for 20 minutes. Centrifuging at 13000rpm for 10 minutes. Discarding the supernatant. Adding 500μL 75% ethanol, allowing it to stand at room temperature for 5 minutes. Centrifuging at 13000rpm for 10 minutes, a white precipitate appears at the bottom of the tube. Repeating the 75% ethanol treatment and centrifugation steps once. Discard the supernatant, aspirate as much as possible, and let stand until the ethanol in the tube evaporates and dries. The white precipitate at the bottom of the tube is DNA. Depending on the size of the precipitate, add 50–100 μL of sterile water to dissolve the DNA.

[0025] like Figure 1 As shown, both male and female Chinese softshell turtles produce one band, while females produce two bands. Therefore, the sex of the Chinese softshell turtle can be determined by detecting the amplified product through electrophoresis.

[0026] Further sequencing of the amplified products, such as... Figure 2 As shown. If only the sequence shown in SEQ ID NO:3 is detected, the Chinese softshell turtle being tested is a male. If the sequences shown in SEQ ID NO:3 and 4 are detected, the Chinese softshell turtle being tested is a female.

[0027] Therefore, it can be seen that the primer pairs, kits and methods provided in the examples can effectively identify the sex of Chinese soft-shelled turtles.

[0028] The above description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.

[0029] The above description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.

Claims

1. A primer pair consisting of a DNA as shown in SEQ ID NO: 1 and a DNA as shown in SEQ ID NO:

2. 2.A kit comprising the primer pair of claim 1 and other reagents required for PCR amplification. 3.The kit of claim 2, wherein the other reagents required for PCR amplification comprise 2 × Phanta Flash Master Mix. 4.A method for identifying the gender of a Chinese soft-shelled turtle, comprising: performing PCR amplification on genomic DNA of a Chinese soft-shelled turtle to be tested using the primer pair of claim 1; and identifying the gender of the Chinese soft-shelled turtle to be tested according to the amplification product; wherein the step of identifying the gender of the Chinese soft-shelled turtle to be tested according to the amplification product comprises: performing electrophoresis detection on the amplification product, if only one band is detected, then the Chinese soft-shelled turtle to be tested is a male individual; and if two bands are detected, then the Chinese soft-shelled turtle to be tested is a female individual. 5.Use of the primer pair of claim 1, the kit of claim 2 or claim 3, or the method of claim 4 in the identification of the gender of a Chinese soft-shelled turtle. ​ ​ ​ ​ ​ ​

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