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

Abstract

The application relates to the technical field of gender identification of Trionychidae, in particular to a primer pair for identifying the gender of Trionychidae, a kit, a method and application. The primer pair comprises DNA as shown in SEQ ID NO: 1 and 2. The kit comprises the primer pair. The method comprises performing PCR amplification on the genomic DNA of Trionychidae by using the primer pair, and identifying the gender of Trionychidae according to the PCR amplification product. The application can extremely simply identify the gender of Chinemys reevesii, Palea steindachneri, Palea steindachneri and Apalone spinifera, and then fast-growing all-male or all-female fry can be produced, which has important application value for aquaculture.

Description

Technical Field

[0001] This application relates to the technical field of sex identification in turtles, specifically to primer pairs, reagent kits, methods, and applications for identifying the sex of turtles. Background Technology

[0002] There are 4 genera and 7 species of common turtles in the family Trionyxe. Among them, the Chinese softshell turtle (Pelodiscus sinensis) and the mountain softshell turtle (Palea steindachneri) have been commercially developed, along with two introduced species: the pearl softshell turtle (Apaloneferox) and the horned softshell turtle (Apalone spinifera). The significant differences in production performance between the sexes of turtles are crucial for their application in aquaculture. Genetic research and breeding practices in turtles often require identifying their sex chromosome composition, and identifying sex-specific molecular markers is a convenient method. Identifying the genetic sex of farmed species early in their growth and development will greatly promote the breeding of asexual seedlings with growth advantages. Therefore, screening for sex-related molecular markers and establishing genetic sex identification techniques are particularly important. Summary of the Invention

[0003] This application utilizes whole-genome resequencing technology to conduct in-depth genome mining of Chinese soft-shelled turtle, mountain soft-shelled turtle, pearl soft-shelled turtle, and horned soft-shelled turtle to identify sex-specific sequences. Based on these female-specific sequences, specific primer pairs, kits, and methods were obtained for the identification of sex in turtles, and to develop sex-specific molecular markers for sex differentiation.

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

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

[0006] Secondly, the embodiments disclose a kit comprising DNA as shown in SEQ ID NO:1 and 2.

[0007] Thirdly, the embodiments disclose a method for identifying the sex of turtles. The method includes: obtaining genomic DNA from a turtle; performing PCR amplification using the primer pair from the first aspect; and identifying the sex of the turtle based on the PCR amplification product.

[0008] Fourthly, 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 turtles. Attached Figure Description

[0009] Figure 1Electrophoresis diagram of Chinese soft-shelled turtle amplified by conventional PCR using primer pairs TY-WF and TY-WR provided for the example.

[0010] Figure 2 Electrophoresis diagram of pearl turtle amplified by conventional PCR using primer pairs TY-WF and TY-WR provided for the example.

[0011] Figure 3 Electrophoresis diagram of horned turtle amplified by conventional PCR using primer pairs TY-WF and TY-WR provided for the example.

[0012] Figure 4 Electrophoresis diagram of *Trionyx sinensis* amplified by conventional PCR using primer pairs TY-WF and TY-WR provided for the example.

[0013] Figure 5 The image shows the detection results of primer pairs TY-WF and TY-WR provided in the example using droplet digital PCR. Note: B02 represents the result of a female Chinese softshell turtle sample, C02 represents the result of a male Chinese softshell turtle sample, A03 represents the result of a female pearl softshell turtle sample, C03 represents the result of a male pearl softshell turtle sample, B04 represents the result of a female horned softshell turtle sample, C04 represents the result of a male horned softshell turtle sample, A05 represents the result of a female mountain softshell turtle sample, D06 represents the result of a male mountain softshell turtle sample, and F11 represents the result of the blank control group. Blue dots represent positive droplets, and gray dots represent negative droplets.

[0014] Figure 6 The image shows droplet bar graphs of the primer pairs TY-WF and TY-WR provided in the examples, using droplet digital PCR for four common turtle species (TY-W-FR). Note: PsF represents the results from female Chinese softshell turtles, PsM represents the results from male Chinese softshell turtles, ZBF represents the results from female pearl softshell turtles, ZBM represents the results from male pearl softshell turtles, JBF represents the results from female horned softshell turtles, JBM represents the results from male horned softshell turtles, SRF represents the results from female mountain softshell turtles, SRM represents the results from male mountain softshell turtles, and B7 represents the control group. The leftmost blue bar represents positive droplets, the middle dark blue bar represents negative droplets, and the rightmost green bar represents the total number of droplets. Detailed Implementation

[0015] 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.

[0016] This application provides primer pairs, reagent kits, methods, and applications for sex identification in typical turtle species exhibiting sexual dimorphism in production traits through comprehensive analysis of their genomic information. It enables extremely simple sex determination of species such as the Chinese soft-shelled turtle, the mountain soft-shelled turtle, the pearl soft-shelled turtle, and the horned soft-shelled turtle, thereby facilitating the production of fast-growing all-male or all-female hatchlings, which has significant application value for aquaculture. Furthermore, this research can also provide technical support for the protection, breeding, and release of endangered wild turtle species such as the mountain soft-shelled turtle.

[0017] The whole-genome sequencing steps generally include: extracting genomic DNA samples from Chinese soft-shelled turtles, giant soft-shelled turtles, pearl soft-shelled turtles, and horned soft-shelled turtles using conventional column centrifugation (Marine Animal Tissue Genome Extraction Kit, Tiangen Biotech Co., Ltd.), and performing quality and concentration checks using 1% agarose gel electrophoresis and a micro spectrophotometer (NanoDrop2000) to meet the requirements of high-throughput sequencing. Sequencing libraries of 350-500 bp fragments were constructed, and paired-end PE150 sequencing was performed using the Illumina Hiseq X Ten platform to obtain clean data for both male and female genomes. Specific library construction steps were followed according to the Novogene NGSDNALibrary Prep Kit manual. The libraries were then sequenced. Based on the sequencing sequences and the reference genome, sex-difference sequences were mined. From 55 designed specific primer pairs, one pair of universal specific primers for sex identification of the four turtle species (Chinese soft-shelled turtle, pearl soft-shelled turtle, horned soft-shelled turtle, and giant soft-shelled turtle) was selected, resulting in primer pair TY-WF (SEQ ID NO:1) and TY-WR (SEQ ID NO:2).

[0018] Based on this, the embodiments disclose a primer pair comprising DNA as shown in SEQ ID NO:1 and 2.

[0019] Examples disclose a kit comprising DNA as shown in SEQ ID NO:1 and 2.

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

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

[0022] In addition, the embodiments also disclose a method for identifying the sex of turtles. The method includes obtaining genomic DNA of the turtle to be tested; performing PCR amplification using primer pairs TY-WF (SEQ ID NO:1) and TY-WR (SEQ ID NO:2); and identifying the sex of the turtle based on the PCR amplification products.

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

[0024] Prepare a PCR amplification reaction system, wherein the PCR amplification reaction system comprises 20 μL of 2×Taq PlusMasterMix 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;

[0025] The PCR amplification reaction system was pre-denatured at 94℃ for 2 min;

[0026] 94℃ denaturation for 30s, 57℃ annealing for 30s, 72℃ extension for 2min, 35 cycles;

[0027] Extend the heat at 72℃ for 5 minutes.

[0028] In some embodiments, the sex of turtles is identified based on PCR amplification products, including electrophoresis detection of the PCR amplification products. If a stable bright band is detected, the turtle is female; if no stable bright band is detected, the turtle is male.

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

[0030] Prepare a droplet digital PCR reaction system, wherein the droplet digital PCR reaction system contains, in 20 μL increments, 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;

[0031] The prepared droplet digital PCR reaction system is added to the special sample slot of the droplet generator to form droplets;

[0032] The generated droplets were transferred to a PCR instrument and activated at 95°C for about 5 minutes, with one cycle.

[0033] Then, the process involves denaturation at 95°C for 30 seconds, annealing at 57°C for 1 minute, and repeating the denaturation and annealing cycle 50 times; followed by stabilization at 4°C for 1 minute, and then stabilization cycle 1 time.

[0034] Cure microdroplets at 98℃ for 10 minutes.

[0035] In some embodiments, the sex of turtles is identified based on PCR amplification products, including: counting the number of positive droplets; if a number of positive droplets is detected, the turtle is female; if no number of positive droplets is detected, the turtle is male.

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

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

[0038] I. Conventional PCR

[0039] Sexually mature Chinese softshell turtle, mountain softshell turtle, pearl softshell turtle, and horned softshell turtle were obtained, and 10 males and 10 females were obtained through dissection. The tail fins of each softshell turtle were cut off, and DNA samples were prepared using the same standard column centrifugation method (Marine Animal Tissue Genome Extraction Kit, Tiangen Biotech Co., Ltd.) according to the instructions.

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

[0041] like Figures 1-4 Electrophoretic images of 10 individuals each of the Chinese softshell turtle, pearl softshell turtle, horned softshell turtle, and mountain softshell turtle after the above steps are shown. In the images, the primer showed no stable bright band in males of the four turtle species, but a stable bright band was observed in females. This demonstrates that the universal and specific primer can effectively identify the sex of the Chinese softshell turtle, pearl softshell turtle, horned softshell turtle, and mountain softshell turtle.

[0042] II. Droplet Digital PCR

[0043] 1. Selecting dyes and reagents

[0044] Commonly used fluorescent dyes include EvaGreen. A premixed reaction solution specifically for ddPCR, containing enzymes, dNTPs, buffer, and magnesium ions, is also prepared. DNA samples from Chinese soft-shelled turtles, mountain soft-shelled turtles, pearl soft-shelled turtles, and horned soft-shelled turtles were extracted using the same method as in the examples above.

[0045] 2. Preparation of the reaction system

[0046] The droplet digital PCR reaction system, in 20 μL volume, contains 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.

[0047] 3. Droplet formation

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

[0049] 4. PCR reaction

[0050] The generated droplets were transferred to a PCR instrument for amplification. The reaction conditions typically included enzyme activation at 95°C for approximately 5 minutes, repeated once; followed by denaturation at 95°C for 30 seconds, annealing at 57°C for 1 minute, repeated approximately 50 times; finally, signal stabilization at 4°C for 1 minute, repeated once. The droplets were then solidified at 98°C for 10 minutes.

[0051] 5. Detection and Data Analysis

[0052] After the PCR reaction, droplets are placed in a droplet reader, and the fluorescence signal value of each droplet is detected by selecting an appropriate fluorescence channel. Based on the distribution of the fluorescence signal, the instrument's accompanying analysis software automatically sets a threshold, or a threshold is manually set, to distinguish between positive and negative droplets. Droplets with values ​​above the threshold are identified as positive droplets containing the target gene, while droplets with values ​​below the threshold are identified as negative droplets.

[0053] Statistics and Calculations: The number of positive droplets was counted. Based on the Poisson distribution principle and the number and proportion of positive droplets, the analysis software automatically calculated the copy number or concentration of the target gene and provided the corresponding 95% confidence interval. Samples that did not undergo PCR reaction were 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, with no positive droplet copies. The detection channels show that the total droplet count in the female turtle DNA detection channel, the male turtle DNA detection channel, and the blank group detection channel are all above 10,000, meeting the requirements for ddPCR detection.

[0056] like Figure 3-6 As shown in the bar chart, the universal primer TY-W-FR detected a total of 11,014 droplets in female Chinese softshell turtles, including 633 positive droplets and 10,381 negative droplets; in female pearl softshell turtles, the total number of droplets detected was 11,919, including 1,012 positive droplets and 10,907 negative droplets; and in female horned softshell turtles, the total number of droplets detected was 13,681, including 1,132 positive droplets and 12,549 negative droplets. The total number of droplets detected in female Chinese softshell turtles was 10739, of which 97 were positive and 10642 were negative. No positive droplets were detected in the four types of male softshell turtles or 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 types of female softshell turtles, but not in male softshell turtles. This result is consistent with the experimental results of agarose gel electrophoresis.

[0057] Therefore, the sex of turtles can be determined by the number of droplets in droplet digital PCR. If a positive droplet count is detected, it indicates that the turtle is female; otherwise, it is male.

[0058] 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 comprising DNA as shown in SEQ ID NO: 1 and 2. 2.A kit comprising DNA as shown in SEQ ID NO: 1 and 2. 3.The kit of claim 2, further comprising 2×Taq Plus MasterMix II. 4.The kit of claim 2, further comprising 2×QX200 ddPCR EvaGreen Supermix. 5.A method for identifying the gender of a Trionychidae, comprising: obtaining genomic DNA of a Trionychidae to be tested; performing PCR amplification using the primer pair of claim 1; and identifying the gender of the Trionychidae according to the PCR amplification product, wherein the Trionychidae is Chinese soft-shelled turtle, Palea steindachneri, Pelochelys cantor, and Apalone spinifera. 6.The method of claim 5, wherein the PCR amplification using the primer pair of claim 1 comprises: preparing a PCR amplification reaction system, wherein the PCR amplification reaction system comprises 20 μL of 2×Taq Plus MasterMix II 10 μL, 0.8 μL of 10 μmol / L of the upstream primer shown in SEQ ID NO: 1, 0.8 μL of 10 μmol / L of the upstream primer shown in SEQ ID NO: 2, and 50 ng of genomic DNA; pre-denaturing the PCR amplification reaction system at 94 ℃ for 2 min; denaturing at 94 ℃ for 30 s, annealing at 57 ℃ for 30 s, and extending at 72 ℃ for 2 min, for 35 cycles; and finally extending at 72 ℃ for 5 min. 7.The method of claim 5, wherein the identification of the gender of the Trionychidae according to the PCR amplification product comprises: performing electrophoresis detection on the PCR amplification product, wherein if a stable bright band is detected, the Trionychidae to be tested is a female individual; and if no stable bright band is detected, the Trionychidae to be tested is a male individual. 8.The method of claim 5, wherein the PCR amplification using the primer pair of claim 1 comprises: preparing a droplet digital PCR reaction system, wherein the droplet digital PCR reaction system comprises 20 μL of 2×QX200 ddPCR EvaGreen Supermix 10 μL, 2 μL of genomic DNA, 0.2 μL of 10 μmol / L of the upstream primer shown in SEQ ID NO: 1, 0.2 μL of 10 μmol / L of the upstream primer shown in SEQ ID NO: 2, and 7.6 μL of water; adding the prepared droplet digital PCR reaction system into a special sample tank of a droplet generator to form droplets; transferring the generated droplets to a PCR instrument for enzyme activation at 95 ℃, and the activation time is about 5 min, and the cycle is 1; then denaturing at 95 ℃ for 30 s, annealing at 57 ℃ for 1 min, and denaturing and annealing for 50 cycles; stabilizing at 4 ℃ for 1 min, and stabilizing for 1 cycle; and solidifying the droplets at 98 ℃ for 10 min. ​ 9. The method of claim 5, wherein the gender of the Trionychidae is identified according to the PCR amplification product, comprising: counting the number of positive microdroplets, and if the number of positive microdroplets is detected, the Trionychidae to be tested is a female individual; and if the number of positive microdroplets is not detected, the Trionychidae to be tested is a male individual.

10. Use of the primer pair of claim 1 and the kit of any one of claims 2-4 in identifying the gender of the Trionychidae, wherein the Trionychidae is Pelodiscus sinensis, Palea steindachneri, Palea nungroshi, and Cycloterra spinosa. ​ ​

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