A DNA fragment used for sex determination in Yangtze sturgeon and its applications

Abstract

This invention belongs to the field of animal molecular genetics technology and relates to a DNA fragment for sex identification of Yangtze sturgeon and its uses. It includes a female Yangtze sturgeon-specific DNA fragment CJX, the nucleotide sequence of which is shown in SEQ ID NO.1. This invention also provides a positive reference DNA fragment R4R12, the nucleotide sequence of which is shown in SEQ ID NO.2. This DNA fragment, as a positive reference fragment, binds to the female Yangtze sturgeon-specific DNA fragment CJX for rapid sex identification of Yangtze sturgeon. This invention can effectively improve the efficiency of Yangtze sturgeon farming, further enhance the rationality of Yangtze sturgeon farming, artificially adjust the male-to-female ratio of farmed Yangtze sturgeon, and more rationally utilize the human, material, and financial resources for Yangtze sturgeon farming.

Description

Technical Field

[0001] This invention belongs to the field of animal molecular genetics technology, and relates to a DNA fragment for sex identification of Yangtze sturgeon and its uses. Background Technology

[0002] The Yangtze sturgeon is a species endemic to the upper reaches of the Yangtze River in my country and has significant scientific value in studying global climate change and fish evolution. In recent years, due to overfishing and dam construction, the Yangtze sturgeon population has drastically declined, making it a critically endangered (CR) species on the verge of extinction. Therefore, protecting the Yangtze sturgeon population is extremely urgent and necessary.

[0003] Most vertebrates are dioecious, exhibiting distinct sexual dimorphism in both morphology and physiology. Sex is a crucial factor determining sexual reproduction. With the development of molecular biology, various types of sex markers have been developed, including microsatellite markers, single nucleotide polymorphisms (SNPs), random amplified polymorphic DNA (MADs), and restriction fragment length polymorphisms (RFLPs). The development and application of sex-specific molecular markers and the identification of sex-determining genes can, on the one hand, improve sex-related economic traits, providing necessary tools for sex-controlled breeding; on the other hand, they also lay the foundation for elucidating the molecular mechanisms of sex determination in fish. Sex molecular markers are important tools for developing sex control technologies. This invention provides a molecular marker for identifying the sex of the Yangtze sturgeon, and an internal control marker used in conjunction with this marker. Using this method, the sex of the Yangtze sturgeon can be accurately identified. Summary of the Invention

[0004] The purpose of this invention is to provide a DNA fragment for sex identification of Yangtze sturgeon and its uses, a method for sex identification of Yangtze sturgeon, and a reagent kit.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a DNA fragment for sex identification of Yangtze sturgeon, comprising a female Yangtze sturgeon-specific DNA fragment CJX, the nucleotide sequence of which is shown in SEQ ID NO.1 of the sequence listing.

[0006] Furthermore, it also includes the positive reference DNA fragment R4R12, whose nucleotide sequence is shown in SEQ ID NO.2 of the sequence listing.

[0007] Furthermore, the present invention provides the application of the DNA fragment described above for sex identification of Yangtze sturgeon in the identification of the sex of Yangtze sturgeon.

[0008] Furthermore, this invention provides a method for identifying the sex of a Yangtze sturgeon. Genomic DNA of the Yangtze sturgeon to be identified is extracted, and DNA fragments CJX and R4R12 are amplified separately. If the amplification results are 334 bp and 153 bp, or if the amplification sequence is the nucleotide sequence shown in SEQ ID NO.1 and SEQ ID NO.2, then the Yangtze sturgeon to be identified is female. If the amplification result is only 153 bp, or if the amplification sequence is only the nucleotide sequence shown in SEQ ID NO.2, then the Yangtze sturgeon to be identified is male.

[0009] Furthermore, the present invention provides a kit for identifying the sex of Yangtze sturgeon, comprising primer pairs for amplifying DNA fragments CJX and R4R12.

[0010] Furthermore, the primers used for the DNA fragment CJX are shown in SEQ ID NO.3 and SEQ ID NO.4; and the primers used for amplifying the DNA fragment R4R12 are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0011] Compared with existing technologies, this invention has the following advantages: It allows for rapid and accurate sex identification of Yangtze sturgeon. It enables the breeding of Yangtze sturgeon that are entirely female, saving breeding space and making rational use of human, material, and financial resources in Yangtze sturgeon farming. Attached Figure Description

[0012] Figure 1 This is a diagram showing the integrity of the Yangtze sturgeon DNA extracted in Example 1. Figure 2 The image shows the sex identification of the Yangtze sturgeon in Example 2; in the image, M represents DL1000. Detailed Implementation

[0013] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The listed embodiments are intended to illustrate the implementation of the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0014] This invention provides a female Yangtze sturgeon-specific DNA fragment CJX and a positive reference DNA fragment R4R12.

[0015] Example 1: Extraction of DNA from Yangtze sturgeon to be identified In April 2021, the inventors of this application collected fin samples from 12 female Yangtze sturgeon that had laid eggs and 12 male Yangtze sturgeon that had laid sperm at the Yichang Experimental Station of the Yangtze Sturgeon Research Institute of China Three Gorges Corporation. DNA was extracted from these 24 Yangtze sturgeon using the following specific extraction method: (1) Take out the preserved fin sample, cut about 0.5 g, put it into a 2 mL sterile centrifuge tube, add 1×TE sterile buffer, and soak at 4℃ for 12 h; (2) Take out the fin tissue, put it into a sterile extraction buffer containing 350ul, mix gently, add 40ul 20% SDS and 10ul 20 mg / mL proteinase K (final concentration of 400ug / mL), and mix well; (3) Incubate at 56℃ for 4 hours, gently shaking the centrifuge tube every half hour to facilitate digestion. If the tissue is difficult to digest, the incubation time can be extended to 8 hours, or digested overnight at 37℃; (4) After the tissue has been completely digested, remove the centrifuge tube, add 300 μL of 6 M NaCl solution, and immediately shake vigorously for 30 seconds; Centrifuge at 12000 rpm for 10 min; (5) Discard the precipitate, pour the supernatant into a new centrifuge tube, centrifuge again under the same conditions, take the supernatant, add an equal volume of pre-cooled isopropanol, and place at 20°C for more than 1 hour. (6) Centrifuge at 4℃ and 12000 rpm for 15 min; (7) Discard the supernatant, wash the precipitate once with 70% ethanol solution, and dry it at 37°C or air dry it naturally; (8) Add 50 μL of sterile double-distilled water to dissolve the DNA. After it is completely dissolved, dilute it to 100 ng / μL and store it in a refrigerator at 4°C for later use.

[0016] The integrity of the extracted DNA was determined using a 1% agarose gel electrophoresis test with DL1000 marker. The results are as follows. Figure 1 As shown.

[0017] Example 2: Identifying the sex of Yangtze sturgeon DNA was extracted from 24 Yangtze sturgeon (12 males and 12 females) for molecular identification. PCR was performed using the primers provided in this invention (Table 1). The PCR amplification system was 25 μL: 3 μL 10×PCR Buffer, 2 μL 2.5 mmol / L dNTPs, 3 μL MgCl2, 1 μL each of forward and reverse primers, 0.5 μL Taq enzyme, 2 μL DNA template, and 10.5 μL ultrapure water. The PCR reaction program was: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, annealing at 30 s, extension at 72℃ for 30 s, 35 cycles; 72℃ extension for 10 min; storage at 4℃. The samples were then subjected to 1% agarose gel electrophoresis for 30 minutes at 100 V. The results were observed on a gel electrophoresis apparatus.

[0018] Table 1 Primer Sequences

[0019] If the amplification result is 334bp and 153bp, or if the amplification sequence is the nucleotide sequence shown in SEQ ID NO:1 and SEQ ID NO:2, then the sex of the Yangtze sturgeon is female. If the amplification result is only 153bp, or if the amplification sequence is only the nucleotide sequence shown in SEQ ID NO:2, then the sex of the Yangtze sturgeon is male.

[0020] Specific results are as follows Figure 2 As shown, the method of this invention identified 12 males and 12 females among 24 Yangtze sturgeon. This result is consistent with the sampling records. This demonstrates that the female-specific DNA fragment CJX provided by this invention can be used for accurate sex identification of Yangtze sturgeon.

Claims

1. A DNA fragment for sex determination of Yangtze sturgeon, characterized in that, It includes the female Yangtze sturgeon-specific DNA fragment CJX, whose nucleotide sequence is shown in SEQ ID NO.1 of the sequence listing.

2. The DNA fragment for sex determination of Yangtze sturgeon according to claim 1, characterized in that: It also includes the positive reference DNA fragment R4R12, whose nucleotide sequence is shown in SEQ ID NO.2 of the sequence listing.

3. The application of the DNA fragment described in claim 1 or 2 in identifying the sex of Yangtze sturgeon.

4. A method for identifying the sex of a Yangtze sturgeon, characterized in that, Genomic DNA was extracted from the Yangtze sturgeon to be identified, and the DNA fragments CJX and R4R12 described in claim 2 were amplified respectively. When the amplification results were 334bp and 153bp, or the amplification results were the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, the Yangtze sturgeon to be identified was female. If the amplification result is only 153bp, or if the amplification result sequence is only the nucleotide sequence shown in SEQ ID NO.2, then the sex of the Yangtze sturgeon is male.

5. A reagent kit for identifying the sex of Yangtze sturgeon, characterized in that: Contains primer pairs for amplifying the DNA fragment of claim 1 or claim 2.

6. The reagent kit according to claim 5, characterized in that: Primers used for amplifying DNA fragment CJX are shown in SEQ ID NO.3 and SEQ ID NO.4; primers used for amplifying DNA fragment R4R12 are shown in SEQ ID NO.5 and SEQ ID NO.6.

Images