Primer pair, kit and method for identifying gender of trionychidae animals and application

Abstract

The invention relates to the technical field of trionychidae sex identification, in particular to a primer pair, a kit and a method for identifying the sex of trionychidae animals and application. The primer pair comprises DNA (deoxyribonucleic acid) as shown in SEQ ID NO: 1 and SEQ ID NO: 2. The kit comprises the primer pair. The method comprises the following steps: carrying out PCR (Polymerase Chain Reaction) amplification on genome DNA (Deoxyribonucleic Acid) of the trionychidae by adopting the primer pair, and identifying the gender of the trionychidae according to a PCR amplification product. According to the method, the genders of the Chinese softshell turtles, the wattle-necked softshell turtles, the apalone ferox and the horn turtles can be simply and conveniently determined, then all-male or all-female fries which grow rapidly can be produced, and the method has important application value for the aquaculture industry.

Description

Technical Field

[0001] The present application relates to the technical field of sex identification of Trionychidae, and in particular to primer pairs, kits, methods and applications for sex identification of Trionychidae animals. Background Art

[0002] There are 7 species in 4 genera of common Trionyx species. Among them, Pelodiscussinensis and Palea steindachneri have been industrially developed, as well as two introduced species, Apaloneferox and Apalone spinifera. The sexes of Trionyx species have very different production performances, which is of great significance for their application in aquaculture. In genetic research and breeding practice of Trionyx species, it is often required to identify their sex chromosome composition. Finding sex-specific molecular markers is a more convenient way to identify them. If the genetic sex of the cultured species is identified in the early stage of its growth and development, it will greatly promote the cultivation process of single-sex seedlings with growth-advantage sex. Therefore, it is particularly important to screen its sex-related molecular markers and establish genetic sex identification technology. Summary of the invention

[0003] This application uses whole genome resequencing technology to conduct in-depth whole-genome mining of Chinese soft-shelled turtle, mountain soft-shelled turtle, pearl soft-shelled turtle and horned soft-shelled turtle to find male and female difference sequences. Based on these female-specific sequences, specific primer pairs, kits and methods are obtained to identify the sex of soft-shelled turtles and develop sex-specific molecular markers for distinguishing males and females.

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

[0005] In a first aspect, the embodiments disclose a primer pair comprising DNA as shown in SEQ ID NO: 1 and 2.

[0006] In a second aspect, the embodiment discloses a kit comprising the DNA shown in SEQ ID NO: 1 and 2.

[0007] In the third aspect, the embodiment discloses a method for identifying the sex of a Trionychidae animal. The method comprises: obtaining genomic DNA of the Trionychidae animal; performing PCR amplification using the primer pair of the first aspect; and identifying the sex of the Trionychidae animal based on the PCR amplification product.

[0008] In a fourth aspect, the embodiments disclose the use of the primer pair of the first aspect and the kit of the second aspect in identifying the sex of a Trionycidae animal. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1This is the electrophoresis diagram of the conventional PCR amplification of Chinese soft-shell turtle using the primer pair TY-WF and TY-WR provided in the example.

[0010] Figure 2 This is the electrophoresis diagram of the conventional PCR amplification of pearl softshell turtle using the primer pair TY-WF and TY-WR provided in the example.

[0011] Figure 3 This is the electrophoresis diagram of conventional PCR amplification of horned softshell turtle using the primer pair TY-WF and TY-WR provided in the example.

[0012] Figure 4 This is the electrophoresis diagram of conventional PCR amplification of Trionyx japonicus using the primer pair TY-WF and TY-WR provided in the example.

[0013] Figure 5 The results of the droplet digital PCR test of the primer pair TY-WF and TY-WR provided in the example. Note: B02 represents the results of female samples of Chinese soft-shelled turtle, C02 represents the results of male samples of Chinese soft-shelled turtle, A03 represents the results of female samples of pearl soft-shelled turtle, C03 represents the results of male samples of pearl soft-shelled turtle, B04 represents the results of female samples of horned soft-shelled turtle, C04 represents the results of male samples of horned soft-shelled turtle, A05 represents the results of female samples of mountain soft-shelled turtle, D06 represents the results of male samples of mountain soft-shelled turtle, and F11 represents the results of the blank control group. Blue dots represent positive droplets, and gray dots represent negative droplets.

[0014] Figure 6 The droplet column chart of the primer pair TY-WF and TY-WR provided in the embodiment by droplet digital PCR of the universal primer TY-W-FR of 3-12 four species of Trionyx japonicus. Note: PsF represents the result of female Chinese soft-shell turtle, PsM represents the result of male Chinese soft-shell turtle, ZBF represents the result of female pearl soft-shell turtle, ZBM represents the result of male pearl soft-shell turtle, JBF represents the result of female horned soft-shell turtle, JBM represents the result of male horned soft-shell turtle, SRF represents the result of female mountain soft-shell turtle, SRM represents the result of male mountain soft-shell turtle, and B7 represents the result of the blank control group. The blue column on the far left represents the positive droplet, the dark blue column in the middle represents the negative droplet, and the green column on the far right represents the total number of droplets. DETAILED DESCRIPTION

[0015] 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.

[0016] This application provides primer pairs, kits, methods and applications for identifying the sex of turtles through comprehensive analysis of the genome information of typical turtles with sexual dimorphism in production traits. It can easily identify the sex of Chinese soft-shelled turtles, mountain soft-shelled turtles, pearl soft-shelled turtles and horned soft-shelled turtles, and then produce fast-growing all-male or all-female seedlings, which has important application value for aquaculture. In addition, this study can also provide technical support for the protection, breeding, proliferation and release of endangered wild turtles such as mountain soft-shelled turtles.

[0017] The whole genome sequencing steps generally include: extracting genomic DNA samples of Chinese soft-shell turtle, mountain soft-shell turtle, pearl soft-shell turtle and horned soft-shell turtle by conventional column centrifugation method (marine animal tissue genome extraction kit, Tiangen Biochemical Technology Co., Ltd.), and using 1% agarose gel electrophoresis and micro-spectrophotometer (NanoDrop2000) for quality and concentration detection to meet the requirements of high-throughput sequencing. Sequencing libraries of 350-500bp fragments were constructed respectively, and double-end (Pair-End) PE150 sequencing was performed using the Illumina Hiseq X Ten platform to obtain clean data for female and male genome sequencing. The library construction steps were specifically referred to the Novogene NGSDNALibrary Prep Kit instructions, and then the library was sequenced. According to the sequencing sequence and the reference genome, the male and female difference sequences were mined, and a pair of universal specific primers for male and female identification of four turtles of the family Chinese soft-shell turtle, pearl soft-shell turtle, horned soft-shell turtle and mountain soft-shell turtle were screened from the designed 55 pairs of specific primers to obtain primer pairs TY-WF (SEQ ID NO: 1) and TY-WR (SEQ ID NO: 2).

[0018] Based on this, the embodiment discloses a primer pair including DNA shown in SEQ ID NO: 1 and 2.

[0019] The embodiment discloses a kit comprising the DNA shown in SEQ ID NO: 1 and 2.

[0020] In some embodiments, the kit is a conventional PCR kit, which also includes 2×Taq Plus Master Mix II.

[0021] In some embodiments, the kit is a droplet digital PCR kit, and the kit also includes 2×QX200ddPCR EvaGreen Supermix.

[0022] In addition, the embodiment also discloses a method for identifying the sex of a Trionychidae animal. The method comprises obtaining genomic DNA of the Trionychidae animal to be tested; performing PCR amplification using primer pair TY-WF (SEQ ID NO: 1) and TY-WR (SEQ ID NO: 2); and identifying the sex of the Trionychidae animal according to the PCR amplification product.

[0023] In some embodiments, PCR amplification is performed using primer pair TY-WF (SEQ ID NO: 1) and TY-WR (SEQ ID NO: 2), comprising:

[0024] A PCR amplification reaction system was prepared, wherein the PCR amplification reaction system included 10 μL of 2×Taq Plus Master Mix II, 0.8 μL of 10 μmol / L upstream primer shown in SEQ ID NO: 1, 0.8 μL of 10 μmol / L upstream primer shown in SEQ ID NO: 2, and 50 ng of genomic DNA in 20 μL;

[0025] The PCR amplification reaction system was pre-denatured at 94°C for 2 minutes;

[0026] Denaturation at 94°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 2 min, for 35 cycles;

[0027] Final extension was performed at 72°C for 5 min.

[0028] In some embodiments, the sex of the turtle family animal is identified based on the PCR amplification product, including performing electrophoresis detection on the PCR amplification product. If a stable bright band is detected, the turtle family animal to be detected is a female individual; if no stable bright band is detected, the turtle family animal to be detected is a male individual.

[0029] In some embodiments, PCR amplification is performed using primer pair TY-WF (SEQ ID NO: 1) and TY-WR (SEQ ID NO: 2), comprising:

[0030] A droplet digital PCR reaction system was prepared, wherein the droplet digital PCR reaction system contained 10 μL of 2×QX200ddPCR EvaGreen Supermix, 2 μL of genomic DNA, 0.2 μL of 10 μmol / L upstream primer shown in SEQ ID NO: 1, 0.2 μL of 10 μmol / L upstream primer shown in SEQ ID NO: 2, and 7.6 μL of water in 20 μL;

[0031] Add the prepared droplet digital PCR reaction system into the dedicated sample slot of the droplet generator to form droplets;

[0032] The generated droplets were transferred to a PCR instrument for enzyme activation at 95°C for about 5 minutes, and the cycle was repeated once;

[0033] Then, denaturation at 95°C for 30s, annealing at 57°C for 1min, denaturation and annealing cycles were repeated 50 times; stabilization at 4°C for 1min, stabilization cycle was repeated once;

[0034] The droplets were cured at 98°C for 10 min.

[0035] In some embodiments, the sex of the turtle family animal is identified based on the PCR amplification product, including: counting the number of positive droplets, if the number of positive droplets is detected, the turtle family animal to be tested is a female individual; if the number of positive droplets is not detected, the turtle family animal to be tested is a male individual.

[0036] In addition, the examples also disclose the use of primer pair TY-WF (SEQ ID NO: 1) and TY-WR (SEQ ID NO: 2) and the above kit in identifying the sex of Trionyx species.

[0037] The present application will be described below in conjunction with more specific embodiments, but this does not constitute a limitation on the implementation methods of the present application.

[0038] 1. Conventional PCR

[0039] Obtained visually mature Chinese soft-shelled turtle, mountain soft-shelled turtle, pearl soft-shelled turtle and horned soft-shelled turtle, and obtained 10 female and male soft-shelled turtles by dissection. The tail fins were cut off respectively, and DNA samples were prepared by conventional column centrifugation method (Marine Animal Tissue Genome Extraction Kit, Tiangen Biochemical Technology Co., Ltd.) according to the operating steps in the instruction manual.

[0040] The total PCR amplification reaction system was 20 μL, including 10 μL of 2×TaqPlus MasterMix II (Nanjing Novozyme Biotechnology Co., Ltd.), 0.8 μL of 10 μmol / L upstream primer (shown in SEQ ID NO: 1), 0.8 μL of 10 μmol / L upstream primer (shown in SEQ ID NO: 2), and 50 ng of DNA sample. The PCR reaction program was: 94°C pre-denaturation for 2 min; 94°C denaturation for 30 s, 57°C annealing for 30 s, 72°C extension for 2 min, 35 cycles; 72°C final extension for 5 min. The PCR amplification products were detected by agarose gel electrophoresis.

[0041] like Figures 1 to 4 The electrophoresis diagram of 10 individuals of Chinese soft-shelled turtle, pearl soft-shelled turtle, horned soft-shelled turtle and mountain soft-shelled turtle after the above steps is shown. In the figure, the primer has no stable bright band in the male of the four soft-shelled turtles, but has a stable bright band in the female. This shows that the universal specific primer can effectively identify the sex of Chinese soft-shelled turtle, pearl soft-shelled turtle, horned soft-shelled turtle and mountain soft-shelled turtle.

[0042] 2. Droplet Digital PCR

[0043] 1. Choose dyes and reagents

[0044] Common fluorescent dyes such as EvaGreen, etc. At the same time, a reaction premix solution dedicated to ddPCR is prepared, including enzymes, dNTPs, buffer, magnesium ions, etc. The DNA samples of Chinese soft-shelled turtle, mountain soft-shelled turtle, pearl soft-shelled turtle and horned soft-shelled turtle obtained by extraction were obtained by the same method as in the above embodiment.

[0045] 2. Prepare the reaction system

[0046] The droplet digital PCR reaction system contained 10 μL of 2×QX200 ddPCR EvaGreen Supermix, 2 μL of DNA template, 0.2 μL of 10 μmol / L downstream primer (shown in SEQ ID NO: 1), 0.2 μL of 10 μmol / L upstream primer (shown in SEQ ID NO: 2) and 7.6 μL of water in 20 μL.

[0047] 3. Droplet generation

[0048] Add the prepared droplet digital PCR reaction system to the dedicated sample slot of the droplet generator and generate droplets according to the instrument's operating instructions. Generally speaking, the instrument will divide each sample reaction system into thousands of nanoliter droplets.

[0049] 4. PCR reaction

[0050] The generated droplets are transferred to a PCR instrument for amplification reaction. The reaction conditions usually include enzyme activation at 95°C for about 5 minutes, one cycle; then denaturation at 95°C for 30 seconds, annealing at 57°C for 1 minute, denaturation and annealing cycles for about 50 times; finally, signal stabilization at 4°C for 1 minute, one stabilization cycle. The droplets are solidified at 98°C for 10 minutes.

[0051] 5. Detection and data analysis

[0052] After the PCR reaction is completed, the droplets are placed in the droplet reader and the appropriate fluorescence channel is selected to detect the fluorescence signal value of each droplet. According to the distribution of the fluorescence signal, the analysis software supporting the instrument automatically sets the threshold or manually sets the threshold to distinguish positive droplets from negative droplets. Droplets above the threshold are judged as positive droplets encapsulating the target gene, and droplets below the threshold are negative droplets.

[0053] Statistics and calculation: Count the number of positive droplets, and use analysis software to automatically calculate the copy number or concentration of the target gene based on the Poisson distribution principle and the number and proportion of positive droplets, and give the corresponding 95% confidence interval. Samples without PCR reaction are used as controls.

[0054] 6. Results

[0055] like Figure 3-6 As shown, the DNA detection channel is consistent with the blank group detection channel, and there is no positive droplet copy. The detection channel shows that the total number of droplets in the female turtle DNA detection channel, the total number of droplets in the male turtle DNA detection channel, and the total number of droplets in the blank group detection channel are all above 10,000, meeting the ddPCR detection requirements.

[0056] like Figure 3-6 As shown in the bar graph, it can be clearly seen that the total number of droplets detected by the universal primer TY-W-FR in female Chinese soft-shelled turtles is 11014, of which the number of positive droplets is 633 and the number of negative droplets is 10381. The total number of droplets detected in female pearl soft-shelled turtles is 11919, of which the number of positive droplets is 1012 and the number of negative droplets is 10907. The total number of droplets detected in female horned soft-shelled turtles is 13681, of which the number of positive droplets is 1132 and the number of negative droplets is 12549. A total of 10739 droplets were detected in female mountain soft-shelled turtles, of which 97 were positive droplets and 10642 were negative droplets. No positive droplets were detected in the four male soft-shelled turtles and the blank control group. The molecular weight of the target gene in the sample was determined more accurately from the perspective of absolute quantification. The experimental results showed that the primers only amplified the target gene fragment in the four female soft-shelled turtles, but not in the male soft-shelled turtles. This result was consistent with the experimental results of the agarose gel electrophoresis result gel map.

[0057] It can be seen from this that the sex of the turtle can be determined by the number of droplets of droplet digital PCR. If a positive number of droplets is detected, it indicates that the turtle to be tested is a female individual, otherwise it is a male individual.

[0058] 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 DNAs shown in SEQ ID NOs: 1 and 2.

2. A kit comprising the DNA shown in SEQ ID NO: 1 and 2.

3. The kit according to claim 2, further comprising 2×Taq Plus MasterMix II.

4. The kit according to claim 2, further comprising 2×QX200 ddPCR EvaGreen Supermix.

5. A method for identifying the sex of a Trionyx species, comprising: Obtaining genomic DNA of the turtle species to be tested; Performing PCR amplification using the primer pair as claimed in claim 1; as well as The sex of turtles was identified based on PCR amplification products.

6. The method according to claim 5, using the primer pair according to claim 1 to perform PCR amplification, comprising: A PCR amplification reaction system was prepared, wherein the PCR amplification reaction system included 10 μL of 2×Taq Plus MasterMix II, 0.8 μL of 10 μmol / L upstream primer shown in SEQ ID NO: 1, 0.8 μL of 10 μmol / L upstream primer shown in SEQ ID NO: 2, and 50 ng of genomic DNA in 20 μL; The PCR amplification reaction system was pre-denatured at 94°C for 2 minutes; Denaturation at 94°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 2 min, for 35 cycles; Final extension was performed at 72°C for 5 min.

7. The method according to claim 5, wherein the sex of the turtle is identified based on the PCR amplification product, comprising: Performing electrophoresis detection on the PCR amplification product, if a stable bright band is detected, the turtle animal to be detected is a female individual; If no stable bright band is detected, the turtle animal to be tested is a male individual.

8. The method according to claim 5, using the primer pair according to claim 1 to perform PCR amplification, comprising: A droplet digital PCR reaction system was prepared, wherein the droplet digital PCR reaction system contained 10 μL of 2×QX200ddPCR EvaGreen Supermix, 2 μL of genomic DNA, 0.2 μL of 10 μmol / L upstream primer shown in SEQ ID NO: 1, 0.2 μL of 10 μmol / L upstream primer shown in SEQ ID NO: 2, and 7.6 μL of water in 20 μL; Add the prepared droplet digital PCR reaction system into the dedicated sample slot of the droplet generator to form droplets; The generated droplets were transferred to a PCR instrument for enzyme activation at 95°C for about 5 minutes, and the cycle was repeated once; Then, denaturation at 95°C for 30s, annealing at 57°C for 1min, denaturation and annealing cycles were repeated 50 times; stabilization at 4°C for 1min, stabilization cycle was repeated once; The droplets were cured at 98°C for 10 min.

9. The method according to claim 5, wherein the sex of the turtle is identified based on the PCR amplification product, comprising: Count the number of positive droplets. If positive droplets are detected, the turtle animal to be tested is a female individual. If no positive droplets are detected, the turtle animal to be tested is a male individual.

10. Use of the primer pair according to claim 1 and the kit according to any one of claims 2 to 4 in identifying the sex of Trionyx species.

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