Primer, kit and method for identifying gender of Chinese softshell turtle and application

Abstract

The invention relates to the technical field of Chinese softshell turtles, in particular to primers, a kit and a method for identifying gender of Chinese softshell turtles and application. The primer pair is used for respectively carrying out PCR (Polymerase Chain Reaction) amplification on genome DNAs (Deoxyribose Nucleic Acid) of male individuals and female individuals of the Chinese softshell turtles, so that male specific fragments and female specific fragments can be respectively obtained. The primer pair, the kit and the method provided by the embodiment of the invention can effectively distinguish female individuals and male individuals of the Chinese softshell turtles.

Description

Technical Field

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

[0002] Chinese soft-shell turtle (Pelodiscus sinensis), also known as soft-shelled turtle, round fish, and turtle, belongs to the class Reptilia, order Testudinata, family Testudinidae, and genus Pelodiscus. The Yellow River strain of Chinese soft-shell turtle is mainly distributed in the Yellow River Basin of my country. It has a large body, wide skirt, yellowish body color, and good growth and reproduction. The Japanese strain of Chinese soft-shell turtle is a purebred Chinese soft-shell turtle introduced from my country to Japan in the early 1960s. It was carefully selected, bred, purified, and rejuvenated. Subsequently, the cultivated variety was introduced from Japan to my country and became an excellent strain of Chinese soft-shell turtle suitable for breeding in my country. The Huaihe strain of Chinese soft-shell turtle is mainly distributed along the Huaihe River Basin in Anhui. The back body color is earthy yellow or yellowish brown, the skirt is wide and thick, and the growth rate is second only to the Japanese strain and the Yellow River strain. As an excellent local species, it is increasingly valued. Summary of the invention

[0003] Based on this, the inventors of the present application sequenced the genome of female and male Chinese soft-shelled turtles and found that there were female-specific fragments between female and male Chinese soft-shelled turtles. The genomic DNA of male Chinese soft-shelled turtles was amplified by PCR using primer pairs (SDR-2-F / SDR-2-R). The results showed that male Chinese soft-shelled turtles amplified one band, while females amplified two bands. This shows that the primer pairs, kits and methods provided in the examples can effectively distinguish female and male Chinese soft-shelled turtles.

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

[0005] In a first aspect, the embodiment discloses a primer pair, including a DNA as shown in SEQ ID NO: 1 and a DNA as shown in SEQ ID NO: 2. The primer pair is used to perform PCR amplification on the genomic DNA of Chinese soft-shell turtle, and the sex of Chinese soft-shell turtle can be identified according to the amplified product.

[0006] In the second aspect, the embodiment discloses a kit, which includes the primer pair described in the second aspect and other reagents for PCR amplification.

[0007] In a third aspect, the embodiment discloses a method for identifying the sex of a Chinese soft-shelled turtle, which comprises performing PCR amplification using the genomic DNA of the Chinese soft-shelled turtle to be tested as a template and identifying the sex of the Chinese soft-shelled turtle according to the amplification product.

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

[0009] Figure 1 The electrophoresis result diagram of the method for identifying the sex of Chinese soft-shelled turtle provided in the embodiment. In the diagram, M is a 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 method for identifying the sex of Chinese soft-shelled turtle provided in the embodiment identifies the sequencing peak diagram of the amplification product present in both male and female individuals.

[0011] Figure 3 The method for identifying the sex of Chinese soft-shelled turtle provided in the embodiment is to identify the sequencing peak diagram of the amplification product unique to female individuals. DETAILED DESCRIPTION

[0012] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0013] In the following experimental steps, unless otherwise specified, all operations were performed in accordance with the methods provided in "Molecular Cloning Experimental Guide" (3rd edition, translated by Huang Peitang et al., Beijing: Science Press, 2002).

[0014] The inventors of the present application sequenced the genome of female and male Chinese soft-shelled turtles and found that there were female-specific fragments between female and male Chinese soft-shelled turtles. The genomic DNA of male Chinese soft-shelled turtles was amplified by PCR using primer pairs (such as the DNA shown in SEQ ID NO: 1 and the DNA shown in SEQ ID NO: 2), and male-female differential fragments were obtained. This shows that the primer pairs, kits and methods provided in the examples can effectively distinguish female and male Chinese soft-shelled turtles.

[0015] The embodiment discloses that the primer pair includes the DNA shown in SEQ ID NO: 1 and the DNA shown in SEQ ID NO: 2. The genomic DNA of Chinese soft-shell turtle is amplified by PCR using the primer pair, and the sex of Chinese soft-shell turtle can be identified according to the amplified product.

[0016] The embodiment discloses a kit, which includes the above primer pair and other reagents for PCR amplification. In some embodiments, the other reagents for PCR amplification include 2×Phanta Flash Master Mix (Vazyme).

[0017] The embodiment discloses a method for identifying the sex of a Chinese soft-shelled turtle, which comprises performing PCR amplification using the genomic DNA of the Chinese soft-shelled turtle to be tested as a template and identifying the sex of the Chinese soft-shelled turtle according to the amplified product.

[0018] In some embodiments, the step of identifying the sex of the Chinese soft-shelled turtle according to the amplified product comprises: performing electrophoresis detection on the amplified product, if only one band is detected, the Chinese soft-shelled turtle to be detected is a male individual, and if two bands are detected, the Chinese soft-shelled turtle to be detected is a female individual.

[0019] In some embodiments, the step of identifying the sex of the Chinese soft-shelled turtle according to the amplification product comprises:

[0020] The amplified product is sequenced. If only the sequence shown in SEQ ID NO: 3 is detected, the Chinese soft-shelled turtle to be tested is a male individual. If the sequences shown in SEQ ID NO: 3 and 4 are detected, the Chinese soft-shelled turtle to be tested is a female individual.

[0021] The embodiments disclose the application of the primer pair, the kit or the method in sex identification of Chinese soft-shell turtle and / or breeding using sex identification.

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

[0023] The genomic DNA of muscle tissue of Chinese soft-shelled turtle was collected, and the primer pairs shown in SEQ ID NO: 4 and 5 were used for PCR amplification. The PCR reaction system was calculated as 25 μL, including: 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 reaction program was: 98°C 30s, 32 cycles (98°C denaturation 10s, 58°C annealing 5s, 72°C extension 5s), 72°C extension 1min. The amplified product was detected by 1.2% agarose gel electrophoresis, and the sex of the Chinese soft-shelled turtle was identified according to the band detected by electrophoresis of the amplified product.

[0024] The step of extracting genomic DNA includes: taking about 0.5g of muscle tissue, cutting it into pieces and putting it into a 1.5mL centrifuge tube. Add 500μL Hombuffer buffer and 80U proteinase K, digest at 55℃ for more than 3h, and shake it upside down every 1h. Add 500μL NaCl (4.5M), add 300μL chloroform, and mix it upside down for 20min. Centrifuge at 13000rpm for 10min. After centrifugation, aspirate 500μL of the supernatant and transfer it to a new tube without aspirating the middle layer. Add 350μL isopropanol, gently invert and mix, and a flocculent precipitate appears, and let it stand for 20min. Centrifuge at 13000rpm for 10min. Discard the supernatant. Add 500μL 75% ethanol and let it stand at room temperature for 5 minutes. Centrifuge at 13000rpm for 10min, and there will be a white precipitate at the bottom of the tube. Repeat the steps of 75% ethanol treatment and centrifugation once. Discard the supernatant, absorb as much as possible, and let it stand until the ethanol in the tube evaporates and 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 in the figure, both male and female Chinese soft-shelled turtles produce one band, while females produce two bands. Thus, the sex identification of Chinese soft-shelled turtles can be achieved by detecting the amplified product through electrophoresis.

[0026] The amplified products were further sequenced, such as Figure 2 If only the sequence shown in SEQ ID NO: 3 is detected, the Chinese soft-shelled turtle to be tested is a male individual. If the sequences shown in SEQ ID NO: 3 and 4 are detected, the Chinese soft-shelled turtle to be tested is a female individual.

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

[0028] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

[0029] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. A primer pair comprising the DNA shown in SEQ ID NO: 1 and the DNA shown in SEQ ID NO:

2.

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

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

4. Methods for identifying the sex of Chinese soft-shelled turtle include: PCR amplification was performed using the genomic DNA of the Chinese soft-shelled turtle to be tested as a template; as well as The sex of the Chinese soft-shelled turtle is identified based on the amplified product.

5. The method according to claim 4, wherein the step of identifying the sex of the Chinese soft-shelled turtle according to the amplified product comprises: The amplified product is subjected to electrophoresis detection. If only one band is detected, the Chinese soft-shelled turtle to be tested is a male individual.

6. The method according to claim 5, wherein the step of identifying the sex of the Chinese soft-shelled turtle according to the amplified product further comprises: If two bands are detected, the Chinese soft-shelled turtle to be tested is a female individual.

7. The method according to claim 5, wherein the step of identifying the sex of the Chinese soft-shelled turtle according to the amplified product comprises: The amplified product is sequenced. If only the sequence shown in SEQ ID NO: 3 is detected, the Chinese soft-shelled turtle to be tested is a male individual.

8. The method according to claim 7, wherein the step of identifying the sex of the Chinese soft-shelled turtle according to the amplified product further comprises: If the sequences shown in SEQ ID NOs: 3 and 4 are detected, the Chinese soft-shelled turtle to be tested is a female individual.

9. Use of the primer pair according to any one of claim 1, the kit according to claim 2 or 3, or the method according to any one of claims 4 to 8 in sex identification of Chinese soft-shell turtle and / or breeding using sex identification.

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