Molecular specific marker for identifying genetic sex of pelochelys bibroni and application of molecular specific marker

Abstract

The invention discloses a molecular marker, a primer group and a kit for identifying gender of pelochelys bibroni and application of the molecular marker, the primer group and the kit. The molecular marker has a nucleotide sequence as shown in SEQ ID NO: 1, stably exists in a pelochelys bibroni female individual, and is deleted in a male individual. On the basis, the invention designs a specific primer group which can be used for detecting the molecular marker through PCR (Polymerase Chain Reaction) amplification. The invention also provides a kit containing the primer group and a non-diagnostic identification method. According to the method, the sex is judged by detecting whether a PCR amplification product exists or not: the sex is female when a specific band appears, and the sex is male when no band exists. The specific molecular target for pelochelys bibroni sex identification is provided for the first time, the problem that early-stage and non-invasive sex identification cannot be conducted in the prior art is solved, and the method has the advantages of being accurate, rapid and easy and convenient to operate and has great significance on artificial breeding and population protection of pelochelys bibroni.

Description

Technical Field

[0001] This application relates to the field of turtle sex identification technology, specifically to molecular markers, primer sets, reagent kits, and their applications for identifying turtle sex. Background Technology

[0002] The softshell turtle (Pelochelys cantorii) is a Class I protected aquatic wild animal in China. Due to habitat destruction and human fishing, its wild population is extremely small and it is endangered. Artificial breeding and restocking of softshell turtles are key measures for the protection of this species. Accurately identifying the sex of individuals is a prerequisite for controlling the sex ratio of the population and optimizing breeding strategies, and is of great significance to its conservation biology research.

[0003] Currently, sex determination in softshell turtles mainly relies on morphological observation and anatomical methods. Morphological methods determine sex by observing external morphological differences such as body size, carapace pattern, and tail characteristics. However, this method is only applicable to sexually mature individuals and is greatly affected by the individual's growth and development stage and environmental factors, resulting in low accuracy. While anatomical methods can directly determine sex, they require the sacrifice of individuals, which seriously conflicts with the endangered conservation needs of softshell turtles and cannot be applied to sex determination of live individuals.

[0004] Molecular marker technology provides a non-invasive and accurate new approach for sex identification in wild animals. Studies have developed molecular markers based on sex-related genes in some turtle and tortoise species, but the genome information of the softshell turtle is scarce, and its sex determination mechanism is not yet clear. Existing molecular markers for turtle and tortoise sex cannot be directly applied to sex identification in the softshell turtle.

[0005] Therefore, there is an urgent need to develop a specific and highly accurate molecular marker and related detection method for sex identification of softshell turtles, in order to solve the problems of narrow applicability, low accuracy and high invasiveness of existing technologies for sex identification of softshell turtles, and to provide technical support for the artificial breeding and wild protection of softshell turtles. Summary of the Invention

[0006] To address the technical challenge of early, accurate, and non-invasive sex identification in softshell turtles using existing technologies, this application provides a molecular marker for sex identification. The sequence of this molecular marker is stably present in female softshell turtles but absent in males. By detecting the presence of this molecular marker sequence in softshell turtles, early, non-invasive sex identification can be achieved quickly and with high accuracy. Based on this core discovery, this application also designs a primer set, reagent kit, and detection method for specifically amplifying this molecular marker to identify the sex of softshell turtles.

[0007] Therefore, this application provides the following technical solution:

[0008] In a first aspect, this application provides a molecular marker for identifying the sex of a softshell turtle, wherein the nucleotide sequence of the molecular marker is as shown in SEQ ID NO:1 or has at least 90%, 95%, 98% or 99% identity with the nucleotide sequence shown in SEQ ID NO:1, and retains its female-specific function.

[0009] Through in-depth genomic research, the applicant has for the first time discovered and identified a DNA fragment with the nucleotide sequence shown in SEQ ID NO:1. This sequence is stably present in the genome of female softshell turtles, but has been confirmed to be absent in the genome of males, and can itself serve as a direct basis for sex determination. Theoretically, by using any method capable of detecting the presence of a specific nucleic acid sequence (e.g., nucleic acid hybridization, high-throughput sequencing, etc.), if this sequence is detected in the genome of the softshell turtle being tested, the individual can be determined to be female; conversely, if it is not detected, it is determined to be male. Therefore, the molecular marker provided in this application provides the most fundamental material basis and detection target for solving the problem of sex determination in softshell turtles, laying the foundation for the development of various subsequent specific identification methods.

[0010] Secondly, this application provides a primer set for detecting the molecular markers described in the first aspect, comprising:

[0011] Yuan_F: 5'-CAGTGATTCCTTCCCCTGAA-3' (SEQ ID NO: 2);

[0012] Yuan_R: 5'-CAGCTGTATCACCCCCAACT-3' (SEQ ID NO: 3).

[0013] This primer set was specifically designed based on the molecular marker (SEQ ID NO:1) described in the first aspect. The upstream primer Yuan_F and the downstream primer Yuan_R specifically anneal to the regions flanking the female-specific sequence, thereby efficiently and specifically amplifying the target fragment in the PCR reaction. PCR amplification using this primer set allows for direct determination of the results using techniques such as agarose gel electrophoresis: the presence of an amplified band of the expected size indicates the presence of the female molecular marker in the sample, classifying it as female; the absence of an amplified band indicates male. This primer set provides a core, dedicated tool for molecular sex identification in softshell turtles.

[0014] Thirdly, this application provides a kit for identifying the sex of softshell turtles, which includes the primer set described in the second aspect.

[0015] In a preferred embodiment of this application, the kit further includes Taq polymerase, a mixture of dNTPs, and PCR buffer.

[0016] In a preferred embodiment of this application, the kit further includes a positive control reagent containing a DNA template with a nucleotide sequence as shown in SEQ ID NO:1.

[0017] Fourthly, this application provides a method for identifying the sex of a softshell turtle, comprising the following steps:

[0018] (1) Extract genomic DNA from the turtles to be tested;

[0019] (2) Using the genomic DNA extracted in step (1) as a template, perform PCR amplification using the primer set described in the second aspect;

[0020] (3) Detect the PCR amplification products from step (2) and determine the sex of the turtle to be tested based on whether a specific amplification band is generated. The determination method is as follows:

[0021] If a specific amplification band of the expected size is generated, the turtle being tested is determined to be female; if the specific amplification band is not generated, the turtle being tested is determined to be male.

[0022] In a preferred embodiment of this application, in step (2), the PCR amplification system (20 μL) is as follows:

[0023] 2*Taq MasterMix (Dye) 10 μL, upstream primer 0.8 μL, downstream primer 0.8 μL, genomic DNA 1 μL, ddH2O 7.4 μL.

[0024] In a preferred embodiment of this application, the PCR amplification procedure in step (2) is as follows:

[0025] Pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30 s, annealing at 63 ℃ for 30 s, extension at 72 ℃ for 1 min, for a total of 35 cycles; final extension at 72 ℃ for 5 min.

[0026] In one embodiment of this application, in step (3), the PCR amplification product is detected by agarose gel electrophoresis, and the specific amplification band of the expected size is about 1113 bp.

[0027] Fifthly, this application provides the application of the molecular markers described in the first aspect, the primer sets described in the second aspect, the kits described in the third aspect, and / or the methods described in the fourth aspect in identifying the sex of softshell turtles and / or in softshell turtle breeding.

[0028] Compared with the prior art, this application has at least the following advantages and effects:

[0029] 1. This application is the first experimentally verified female-specific molecular marker directly linked to the sex of the softshell turtle. This molecular marker is stably present in females and completely absent in males, making it a direct and reliable molecular target for sex determination, fundamentally solving the technical challenge of sex identification in softshell turtles.

[0030] 2. This application identifies the sex of softshell turtles at the DNA level, unaffected by physiological or environmental factors such as individual age, season, and health status. The marker is amplified by PCR, and the results are simple and intuitive to interpret – the presence of an amplified band indicates female, and the absence of a band indicates male. This avoids subjective misjudgment from morphological observation and the fluctuations in hormone testing, achieving an accuracy rate close to 100%.

[0031] 3. The turtle sex identification method provided in this application does not rely on secondary sexual characteristics and can perform early sex identification on individuals at any growth stage, such as juveniles and subadults. It can be completed with only a small amount of sample, achieving minimal harm and maximum protection for rare and endangered animals, and completely overcoming the stress, infection and mortality risks brought about by invasive methods such as abdominal exploration.

[0032] 4. The sex identification process for softshell turtles provided in this application is simple and can be operated by ordinary laboratory personnel after simple training. It does not require expensive and complicated equipment, which greatly reduces the identification cost and technical threshold. It is especially suitable for large-scale promotion and application in grassroots conservation units and breeding farms.

[0033] 5. The method for sex identification of softshell turtles provided in this application offers an unprecedented technical tool for conservation and management work such as scientific pairing, optimization of population genetic structure, and formulation of release plans in the artificial breeding of softshell turtles. It will greatly improve the efficiency and scientific nature of conservation work and has significant and far-reaching implications for saving and revitalizing wild softshell turtle populations. Attached Figure Description

[0034] Figure 1 The agarose gel electrophoresis image of the PCR amplification products provided in the embodiments of this application shows the PCR amplification results of 16 samples. Among them, female samples (1, 2, 7, 12, 13, 14, 16, 18) showed specific bands at positions 1,113 bp, while male samples (3, 4, 5, 6, 8, 9, 10, 11) showed no amplification bands. M is the DM2000 DNA Marker. Detailed Implementation

[0035] 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.

[0036] The materials used in the following embodiments are not limited to those listed below, and other similar materials may be used instead. Unless otherwise specified, the instruments shall be used under conventional conditions or as recommended by the manufacturer. Those skilled in the art should have relevant knowledge of the use of conventional materials and instruments.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this application pertains. Before a detailed description of this application, the following definitions are provided to better understand it.

[0038] In this application, unless the context clearly indicates otherwise, the terms "comprising," "including," "containing," "having," etc., shall be understood as open-ended, meaning "including but not limited to." For example, when referring to "a kit comprising A and B," it means that A and B are essential, but the kit may also contain other unspecified components such as C and D.

[0039] To better understand this teaching and without limiting its scope, all figures and other numerical values ​​used in the specification and claims to express quantities, percentages, or proportions should, in all cases, be understood to be modified by the term "about." Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximate values ​​that may vary depending on the desired properties sought. At a minimum, each numerical parameter should be interpreted based at least on the reported significant figures and by applying common rounding techniques.

[0040] This application provides a molecular marker for identifying the sex of softshell turtles, a primer set based on the marker, a kit, a method for identifying the sex of softshell turtles, and its application. The molecular marker is a specific DNA fragment present in the genome of female softshell turtles but missing in males, and its nucleotide sequence is shown in SEQ ID NO:1 below:

[0041]

[0042] Based on this, this application presents a primer set designed to specifically amplify the molecular marker. The sequence of the forward primer Yuan_F is shown in SEQ ID NO:2 (5'-CAGTGATTCCTTCCCCTGAA-3'), and the sequence of the reverse primer Yuan_R is shown in SEQ ID NO:3 (5'-CAGCTGTATCACCCCCAACT-3'). Using this primer set for polymerase chain reaction (PCR), the sex of the softshell turtle can be directly determined by detecting the presence or absence of the amplification product: the appearance of an amplification band of the expected size indicates female, while the absence of a band indicates male.

[0043] To translate this method into a stable and convenient product, embodiments of this application further provide a kit for identifying the sex of softshell turtles. This kit contains at least the aforementioned specific primer set and preferably includes a complete set of reagents required for PCR amplification, such as DNA polymerase, dNTPs, buffer solutions, etc., as well as a positive control and nucleic acid dyes for result interpretation.

[0044] Accordingly, this application further provides a method for identifying the sex of a softshell turtle for non-diagnostic purposes. The core steps include: obtaining a biological sample from the individual softshell turtle to be tested and extracting genomic DNA; using the extracted genomic DNA as a template, performing PCR amplification using the aforementioned primer set; performing electrophoresis detection on the PCR product; and determining the sex based on the detection results. Specifically, the determination criteria are: if the electrophoresis result shows an amplified band of the expected size (1113 bp), the turtle is determined to be female; if no band is found, the turtle is determined to be male.

[0045] The above technical solution and its achieved technical effects will be described in detail below through more specific embodiments.

[0046] Example: PCR Validation of Sex-Specific Molecular Markers in Turtle

[0047] 1. Experimental Samples

[0048] To verify the specificity of the molecular markers and primer sets provided in this application, this embodiment selected 16 individuals of known physiological sex (all from Wanlvyuan Ecological Breeding Co., Ltd., Gaoming District, Foshan City, with a weight of over 10 kg, and whose physiological sex could be clearly distinguished by tail length) as test samples, including 8 females and 8 males. Detailed sample information is shown in Table 1 below.

[0049] Table 1 Sample Information

[0050]

[0051] 2. Specific primer design and synthesis

[0052] Based on the female-specific sequences obtained through screening, PCR verification primers were designed using Primer3 version 4.1.0 software (web link: https: / / bioinfo.ut.ee / primer3-0.4.0 / ). The specific primer sequence information is as follows:

[0053] Upstream primer Yuan_F: 5'-CAGTGATTCCTTCCCCTGAA-3' (SEQ ID NO:2);

[0054] Downstream primer Yuan_R: 5'-CAGCTGTATCACCCCCAACT-3' (SEQ ID NO:3);

[0055] Amplified fragment length: 1113 bp; primer annealing temperature: 63 ℃. The primers were purified to a concentration of 10 μM.

[0056] 3. Genomic DNA extraction

[0057] Genomic DNA was extracted using the general-purpose column-based genomic DNA extraction kit (catalog number: CW2298M) from Jiangsu Kangwei Century Technology Co., Ltd., with the specific operating procedures strictly following the kit instructions. The extracted DNA concentration and purity were measured using a NanoDrop nucleic acid and protein analyzer, requiring an A260 / A280 ratio between 1.8 and 2.0 and a concentration ≥50 ng / μL. Qualified DNA samples were stored at -20 ℃ for later use.

[0058] 4. PCR amplification

[0059] 4.1 Reagents and Consumables

[0060] DNA polymerase: 2×Taq MasterMix (Dye) (purchased from Jiangsu Kangwei Century Technology Co., Ltd., product number: CW0682L), containing Taq DNA polymerase, dNTPs and buffer reagents.

[0061] Primers: Yuan_F and Yuan_R (10 μM).

[0062] Template DNA: Genomic DNA from each sample.

[0063] 4.2 PCR reaction system

[0064] Prepare a 20 μL PCR reaction system. The specific composition is shown in Table 2 below.

[0065] Table 2 PCR reaction system

[0066]

[0067] The PCR reaction conditions are shown in Table 3 below.

[0068] Table 3 PCR reaction conditions

[0069]

[0070] 5. Electrophoretic detection and analysis

[0071] Mix 5 μL of PCR product with 1 μL of 6× Loading Buffer and spot the mixture onto a 1% agarose gel (containing 0.5 μg / mL EB or GelRed nucleic acid dye). Use the DM2000 DNA Marker (Jiangsu Kangwei Century Biotechnology Co., Ltd., catalog number: CW0632M) as the molecular weight standard and perform electrophoresis at a constant voltage of 180 V for 30 minutes. After electrophoresis, observe and photograph the gel using a gel imaging system.

[0072] The DM2000 DNA Marker contains six standard fragments: 2,000 bp, 1,000 bp, 750 bp, 500 bp, 250 bp, and 100 bp. The 750 bp band has a concentration of approximately 90 ng and appears as a bright band, while the other bands have a concentration of approximately 30 ng.

[0073] 6. Result Judgment Criteria

[0074] Positive result (female): A clear, specific band appeared at the 1,113 bp position, corresponding to the marker position.

[0075] Negative result (male): No visible bands.

[0076] Invalid results: If the positive control shows no band or the negative control shows a band, the experiment must be repeated.

[0077] 7. Final Experimental Results

[0078] PCR amplification was performed on 16 samples, and the amplification products were subjected to agarose gel electrophoresis. Figure 1 The electrophoresis results are shown. From Figure 1 The statistical results of PCR amplification are shown in Table 4 below.

[0079] Table 4 Statistical analysis of PCR amplification results

[0080]

[0081] Note: "1" indicates a positive result, with a clear target band; "0" indicates a negative result, with no band.

[0082] from Figure 1As shown in Table 4, all eight female samples exhibited a single, clear, and specific amplification band at the 1,113 bp position, consistent with the expected fragment size; none of the eight male samples showed any amplification band. The identification results of 16 samples with known sex in this embodiment were completely consistent with the physiological sex, achieving an accuracy rate of 100%.

[0083] In summary, this application provides molecular markers for identifying the sex of softshell turtles and establishes a PCR method for sex identification based on female-specific molecular markers. The designed primer pair Yuan_F / Yuan_R exhibits excellent sex specificity and amplification stability, accurately distinguishing between male and female softshell turtle individuals. This method is simple to operate, and the results are clear and easy to interpret, providing a robust and reliable implementation plan for early, non-invasive, and accurate sex identification of softshell turtles. This method provides a reliable molecular tool for sex identification, artificial breeding, population genetic structure analysis, and conservation biology research of softshell turtles.

[0084] The present application has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the embodiments above are only for the purpose of helping to understand the present application and its core ideas. It should be noted that those skilled in the art can make several improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A molecular marker for identifying the sex of a softshell turtle, said molecular marker having a nucleotide sequence as shown in SEQ ID NO:1 or having at least 90%, 95%, 98% or 99% identity with the nucleotide sequence shown in SEQ ID NO:1, and retaining its female-specific function.

2. A primer set for detecting the molecular marker of claim 1, comprising: Yuan_F: Nucleotide sequence as shown in SEQ ID NO:2; Yuan_R: The nucleotide sequence is shown in SEQ ID NO:

3.

3. A kit for identifying the sex of a softshell turtle, comprising the primer set as described in claim 2.

4. The kit according to claim 3 further includes Taq polymerase, a mixture of dNTPs, and PCR buffer.

5. The kit according to claim 4, further comprising a positive control reagent containing a DNA template with a nucleotide sequence as shown in SEQ ID NO:

1.

6. A method for identifying the sex of a softshell turtle, comprising the following steps: (1) Extract genomic DNA from the turtles to be tested; (2) Using the genomic DNA extracted in step (1) as a template, perform PCR amplification using the primer set described in claim 2; (3) Detect the PCR amplification products from step (2) and determine the sex of the turtle to be tested based on whether a specific amplification band is generated. The determination method is as follows: If a specific amplification band of the expected size is generated, the turtle being tested is determined to be female; if the specific amplification band is not generated, the turtle being tested is determined to be male.

7. According to claim 6, in step (2), the PCR amplification system is as follows: 2*Taq MasterMix (Dye) 10 μL, upstream primer 0.8 μL, downstream primer 0.8 μL, genomic DNA 1 μL, ddH2O 7.4 μL.

8. According to the method of claim 5, in step (2), the PCR amplification procedure is as follows: Pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30 s, annealing at 63 ℃ for 30 s, extension at 72 ℃ for 1 min, for a total of 35 cycles; final extension at 72 ℃ for 5 min.

9. According to the method of claim 5, in step (3), the PCR amplification product is detected by agarose gel electrophoresis, and the specific amplification band of the expected size is about 1113 bp.

10. The use of the molecular marker of claim 1, the primer set of claim 2, the kit of any one of claims 3 to 5, and / or the method of any one of claims 6 to 9 in identifying the sex of softshell turtles and / or in softshell turtle breeding.

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