Primers, kit and method for identifying gender of chinese softshell 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] This application relates to the field of Chinese soft-shelled turtle technology, specifically to primers, reagent kits, methods, and applications for identifying the sex of Chinese soft-shelled turtles. Background Technology

[0002] The Chinese softshell turtle (Pelodiscus sinensis), also known as the turtle, is a species belonging to the class Reptilia, order Testudines, family Trionychidae, and genus Pelodiscus. The Yellow River strain of the Chinese softshell turtle is mainly distributed in the Yellow River basin of my country. It is large, has a wide skirt, and a slightly yellowish body color, exhibiting excellent growth and reproductive performance. The Japanese strain of the Chinese softshell turtle was introduced from my country to Japan in the early 1960s as a purebred species. Through careful selection, purification, and rejuvenation, this bred variety was subsequently introduced back to my country from Japan, becoming a superior strain adapted for aquaculture in my country. The Huai River strain of the Chinese softshell turtle is mainly distributed along the Huai River basin in Anhui Province. Its back is yellowish-brown or yellowish-brown, with a wide and thick skirt. Its growth rate is second only to the Japanese and Yellow River strains, and it is increasingly valued as a superior local species. Summary of the Invention

[0003] Based on this, the inventors of this application performed genome sequencing on female and male Chinese softshell turtles and discovered female-specific fragments between them. Using primer pairs (SDR-2-F / SDR-2-R), PCR amplification of the genomic DNA of male Chinese softshell turtles was performed. The results showed that male Chinese softshell turtles amplified one band, while females amplified two bands. This demonstrates that the primer pairs, kits, and methods provided in the examples can effectively distinguish between female and male Chinese softshell turtles.

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

[0005] In one aspect, the embodiments disclose primer pairs, including DNA as shown in SEQ ID NO:1 and DNA as shown in SEQ ID NO:2. Using these primer pairs to perform PCR amplification of the genomic DNA of the Chinese soft-shelled turtle, the sex of the turtle can be identified based on the amplification products.

[0006] Secondly, the embodiments disclose a kit. The kit includes the primer pairs described in the second aspect and other reagents for PCR amplification.

[0007] Thirdly, the embodiments disclose a method for identifying the sex of the Chinese softshell turtle. This method includes PCR amplification using the genomic DNA of the Chinese softshell turtle to be tested as a template, and identifying the sex of the Chinese softshell turtle based on the amplification product.

[0008] Fourthly, the embodiments disclose the application of the above primer pairs, the above kits, or the above methods in sex identification of Chinese soft-shelled turtles and / or in breeding using sex identification. Attached Figure Description

[0009] Figure 1 Electrophoresis results of the method for identifying the sex of Chinese soft-shelled turtles provided in the embodiment. In the figure, M is the marker, lanes 1-6 are the results of male Chinese soft-shelled turtles, and lanes 7-12 are the results of female Chinese soft-shelled turtles.

[0010] Figure 2 The sequencing peak diagram of the amplified product present in both male and female individuals is provided in the example method for identifying the sex of Chinese soft-shelled turtles.

[0011] Figure 3 The sequencing peak diagram of the amplification product unique to female individuals is shown in the embodiment of the method for identifying the sex of Chinese soft-shelled turtles. Detailed Implementation

[0012] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Reagents not specifically described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0013] In the following experimental procedures, unless otherwise specified, all operations shall be performed in accordance with the methods provided in Molecular Cloning: A Laboratory Manual (3rd Edition, translated by Huang Peitang et al., Beijing: Science Press, 2002).

[0014] The inventors of this application performed genome sequencing on female and male Chinese softshell turtles and discovered female-specific fragments between them. Using primer pairs (such as the DNA shown in SEQ ID NO:1 and SEQ ID NO:2), PCR amplification of the genomic DNA of male Chinese softshell turtles yielded sex-differenced fragments. This demonstrates that the primer pairs, kits, and methods provided in this embodiment can effectively distinguish between female and male Chinese softshell turtles.

[0015] The embodiments disclose that the primer pair includes DNA as shown in SEQ ID NO:1 and DNA as shown in SEQ ID NO:2. Using the primer pair to perform PCR amplification of the genomic DNA of the Chinese soft-shelled turtle, the sex of the turtle can be identified based on the amplification products.

[0016] The embodiments disclose a kit comprising the primer pairs described above and other reagents for PCR amplification. In some embodiments, the other reagents for PCR amplification include 2×Phanta Flash Master Mix (Vazyme).

[0017] The embodiments disclose a method for identifying the sex of the Chinese softshell turtle. The method includes PCR amplification using the genomic DNA of the Chinese softshell turtle to be tested as a template, and identifying the sex of the Chinese softshell turtle based on the amplification product.

[0018] In some embodiments, the step of identifying the sex of the Chinese softshell turtle based on the amplification product includes: performing electrophoretic detection on the amplification product; if only one band is detected, the Chinese softshell turtle is male; if two bands are detected, the Chinese softshell turtle is female.

[0019] In some embodiments, the step of identifying the sex of the Chinese softshell turtle based on the amplification product includes:

[0020] The amplified product was sequenced. If only the sequence shown in SEQ ID NO:3 was detected, the tested Chinese softshell turtle was male. If the sequences shown in SEQ ID NO:3 and 4 were detected, the tested Chinese softshell turtle was female.

[0021] The examples disclose the application of the above primer pairs, the above kits, or the above methods in sex identification of Chinese soft-shelled turtles and / or in breeding using sex identification.

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

[0023] Genomic DNA was collected from muscle tissue of individual Chinese soft-shelled turtles and amplified by PCR using primer pairs shown in SEQ ID NO:4 and 5. The PCR reaction volume was 25 μL, containing: 1 μL (100 ng / μL) genomic DNA, 12.5 μL 2×PhantaFlash Master Mix (Vazyme, Catalog#:P510), 1 μL upstream primer, 1 μL downstream primer, and 9.5 μL ddH2O. The PCR program was: 98℃ for 30 s, 32 cycles (98℃ denaturation for 10 s, 58℃ annealing for 5 s, 72℃ extension for 5 s), followed by a final extension at 72℃ for 1 min. The amplified products were detected by 1.2% agarose gel electrophoresis, and the sex of the Chinese soft-shelled turtles was identified based on the bands detected by electrophoresis.

[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 80U 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 DNA shown in SEQ ID NO:1 and DNA shown in SEQ ID NO:

2.

2. A kit comprising the primer pair of claim 1 and other reagents for PCR amplification.

3. The kit according to claim 2, wherein the other reagents for PCR amplification include 2×Phanta FlashMaster Mix.

4. A method for identifying the sex of the Chinese soft-shelled turtle, comprising: Using the genomic DNA of the Chinese soft-shelled turtle as a template, PCR amplification was performed using the primer pair described in claim 1; The PCR amplification products are tested. If only one band is detected, the Chinese softshell turtle being tested is male; if two bands are detected, the Chinese softshell turtle being tested is female.

5. The application of the primer pair of claim 1, or the kit of claim 2 or 3, in sex identification of Chinese soft-shelled turtles.

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