Gender-specific snp molecular marker of mauremys sinensis and method for identifying genetic gender

Abstract

This invention belongs to the field of marker-assisted breeding technology, and particularly relates to sex-specific SNP molecular markers and genetic sex identification methods for pearl softshell turtles. The SNP molecular marker is located at the 128th base of the nucleotide sequence shown in SEQ ID NO. 3 and / or 4. When the 128th base is the AA genotype, the tested pearl softshell turtle is male; when it is the AG genotype, the tested pearl softshell turtle is female. The SNP molecular markers provided by this invention offer an important molecular tool for sex identification and sex determination mechanism research. They can transform the determination of the sex trait of a pearl softshell turtle into the determination of the molecular marker genotype, which is beneficial for sex identification in the early stages of pearl softshell turtle growth. This invention has advantages such as speed and efficiency, short sex detection cycle, high throughput, minimal interference from environmental factors, high accuracy and reliability of detection results, and has broad application prospects and social significance in the field of pearl softshell turtle sex identification.

Description

Technical Field

[0001] This invention relates to the field of molecular marker-assisted breeding technology, and in particular to a sex-specific SNP molecular marker and genetic sex identification method for pearl turtles. Background Technology

[0002] The pearl softshell turtle (Apalonef erox), also known as the Florida softshell turtle or the American mountain softshell turtle, belongs to the class Reptilia, order Chelonia, family Trionychidae, and genus Palea. Native to the southeastern United States, it is characterized by its large size, thick skirt, high meat yield, rich nutrition, and delicious taste. Its nutritional and medicinal value surpasses that of the Chinese softshell turtle. Furthermore, this species is aesthetically pleasing, grows rapidly, is highly adaptable, has few diseases, and is easy to raise. Whether for farming or as an ornamental turtle, it has significant development value and is a promising turtle species for artificial breeding in recent years.

[0003] The economic value of pearl softshell turtles differs significantly between sexes. In terms of growth, females grow faster and reach sexual maturity at a larger size, while males grow slower, exhibiting significant sexual dimorphism. Furthermore, female pearl softshell turtles, due to their continuous egg-laying capacity, are the core resource for breeding and are crucial for maintaining the reproductive cycle of the farmed population. Therefore, in large-scale pearl softshell turtle farming, if genetic sex can be identified immediately after hatching, targeted breeding of male and female individuals can be achieved early in the hatchling stage. This promotes the cultivation of single-sex hatchlings with growth advantages, significantly improving the efficiency and profitability of the farming industry while reducing costs.

[0004] Currently, there is very little literature on pearl softshell turtles, and early molecular identification techniques are still lacking. Traditional visual identification methods are only applicable to sexually mature individuals, with obvious sex differences only appearing after one winter's age. This method suffers from long identification cycles, high costs, and a high risk of errors. Histological methods identify sex through gonadal tissue, but this requires dissection and collection of gonadal tissue, which is highly invasive, can easily lead to individual death, and cannot efficiently process large batches of samples from multiple populations.

[0005] Using molecular markers closely linked to genetic sex, molecular marker sites in candidate individuals can be identified and selected through DNA extraction and PCR detection. This allows for rapid sex determination at various developmental stages (especially the embryonic or juvenile stages), providing a convenient, efficient, low-cost, and large-scale early sex determination method. Currently, molecular sex determination technology has been studied for the Chinese softshell turtle (Trionyx sinensis), whose sex determination mechanism is determined by the ZW gene, and genome sequencing information is available for comparison and screening of specific sequences on the ZW gene. However, genome sequencing of the pearl softshell turtle (Trionyx sinensis) has not yet been conducted, and online databases such as NCBI lack its complete genome sequence, making the development of molecular sex determination technology for the pearl softshell turtle particularly difficult. Therefore, exploring the sex determination mechanism of the pearl softshell turtle, identifying sex-specific molecular markers, and establishing early molecular sex determination technology will provide a research foundation for sex-controlled breeding of the pearl softshell turtle, and is of great significance for reducing the cost of pearl softshell turtle farming and improving its economic benefits. Summary of the Invention

[0006] To address the lack of sex-specific molecular markers for pearl turtles in existing technologies and the problems of long identification cycles, high costs, low accuracy, and / or inefficiency in handling large batches and multi-sample populations in traditional sex identification methods, this invention provides a sex-specific SNP molecular marker for pearl turtles. This provides an important molecular tool for sex identification and sex determination mechanism research in pearl turtles. By detecting the genotype of this SNP molecular marker, early sex prediction and identification can be performed at the genetic level, offering advantages such as speed, efficiency, short detection cycle, high throughput, minimal interference from environmental factors, high accuracy, and good reliability. Therefore, this invention provides the application of this SNP molecular marker, its detection primer pairs, or detection kits in pearl turtle sex identification. This invention is specifically achieved through the following technical solutions:

[0007] The first aspect of this invention provides the application of a sex-specific SNP molecular marker for pearl turtles, or its detection primer pair or detection kit, in the sex identification of pearl turtles; the SNP molecular marker is located at the 128th base of the nucleotide sequence shown in SEQ ID NO.3 and / or 4, exhibits A / G polymorphism, and has AG and AA genotypes;

[0008] Specifically, when the 128th base is of the AA genotype, the individual being tested is a male; when the 128th base is of the AG genotype, the individual being tested is a female.

[0009] The second aspect of the present invention provides a detection primer pair for detecting sex-specific SNP molecular markers of pearl turtles, wherein the SNP molecular markers are located at the 128th base of the nucleotide sequence shown in SEQ ID NO. 3 and / or 4, exhibit A / G polymorphism, and have AG and AA genotypes;

[0010] The detection primer pair includes an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2.

[0011] A third aspect of the present invention provides a detection kit comprising the detection primer pairs described above.

[0012] Furthermore, the detection kit also includes PCR amplification reagents, which include Taq DNA polymerase, dNTPs, and buffer reagents.

[0013] The fourth aspect of this invention provides a method for identifying the sex of a pearl turtle, comprising the following steps:

[0014] Genomic DNA was extracted from the individual pearl softshell turtle to be tested;

[0015] The genomic DNA was amplified by polymerase chain reaction using primer pairs as shown in SEQ ID NO.1-2 to obtain amplification products;

[0016] The genotype of the amplification product at position 128 is detected, and the sex of the individual pearl turtle to be tested is determined based on the genotype.

[0017] The amplified product has AA and AG genotypes at position 128. When it is AA genotype, the individual to be tested is determined to be male, and when it is AG genotype, the individual to be tested is determined to be female.

[0018] Furthermore, if the amplification product is detected to include only the nucleotide sequence shown in SEQ ID NO.3, the individual to be tested is determined to be male; if the amplification product is detected to include both the nucleotide sequences shown in SEQ ID NO.3 and 4, the individual to be tested is determined to be female.

[0019] Furthermore, the PCR amplification reaction system, in 40 μL increments, comprises: 20 μL of 2×Taq Master Mix, 1.6 μL of upstream primer, 1.6 μL of downstream primer, 1 μL of genomic DNA, and 15.8 μL of deionized water.

[0020] Furthermore, the PCR amplification reaction program includes: 94 °C pre-denaturation for 3 min, 94 °C denaturation for 30 s, 50 °C annealing for 30 s, 72 °C extension for 15 s, for a total of 35 cycles; running at 72 °C for 5 min; and storing the amplification reaction solution at 4 °C for later use.

[0021] Furthermore, the genotype was detected using Sanger sequencing.

[0022] The advantages and positive effects of this invention are as follows:

[0023] The SNP molecular markers closely linked to the genetic sex traits of pearl softshell turtles provided by this invention offer an important molecular tool for sex identification and sex determination mechanism research. They can transform the determination of individual sex traits in pearl softshell turtles into the determination of SNP molecular marker genotypes, enabling sex identification at all growth stages of pearl softshell turtles. This facilitates early targeted breeding in the pearl softshell turtle farming industry and offers advantages such as speed and efficiency, short sex detection cycle, high throughput, minimal interference from environmental factors, high accuracy, and good reliability. It has broad application prospects and social significance in the field of pearl softshell turtle sex identification, and is of great importance to improving farming efficiency and overall yield, and promoting the high-quality and sustainable development of the pearl softshell turtle farming industry. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is an agarose gel electrophoresis image of the amplification products of the sex-specific SNP molecular markers of the pearl turtle in an embodiment of the present invention;

[0026] Figure 2 This is a sequencing peak diagram of the sex-specific SNP molecular marker amplification product of male pearl softshell turtles in an embodiment of the present invention;

[0027] Figure 3 This is a sequencing peak diagram of the sex-specific SNP molecular marker amplification product of female pearl softshell turtles in an embodiment of the present invention;

[0028] Figure 4 This is a sequencing comparison diagram of different genotypes of sex-specific SNP molecular markers in male and female pearl softshell turtles according to an embodiment of the present invention. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0030] Based on the information contained herein, various changes to the precise description of the invention can be readily made by those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of the invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention. In fact, various modifications to embodiments of the invention that will be apparent to those skilled in the art or related fields are covered within the scope of the appended claims.

[0031] To better understand the invention and not to limit its scope, all figures and other numerical values ​​used in this invention to indicate amounts, percentages, or other quantities should, in all cases, be understood to be modified by the word "approximately." Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods.

[0032] The terms “comprising,” “including,” “containing,” “having,” and similar words are non-restrictive and can include other steps and other components that do not affect the result. The term “and / or” should be considered as a specific disclosure of each of the two specified features or components, with or without the other. For example, “A and / or B” is considered to include (i) A, (ii) B, and (iii) A and B.

[0033] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below.

[0034] Single nucleotide polymorphism (SNP) molecular markers refer to DNA sequence polymorphisms caused by variations in a single nucleotide at the genomic level, including single-base transversions, transitions, insertions, and deletions. They are the most numerous and widely distributed molecular markers in the genome, possessing advantages such as ease of genotyping, good genetic stability, and ease of automation and batch processing, and are considered the most valuable next-generation genetic markers. Developing novel SNP molecular markers that are specifically associated with or linked to the sex of pearl softshell turtles will provide important molecular tools for in-depth research on the sex determination mechanisms of pearl softshell turtles and other turtle species, offering effective means of early sex identification in pearl softshell turtles and providing strong support for the sustainable development of pearl softshell turtle aquaculture.

[0035] Since the pearl softshell turtle lacks complete whole-genome information, this invention first performed whole-genome resequencing on 20 randomly selected pearl softshell turtles. The raw high-throughput sequencing data was then quality-filtered and assembled to obtain genomic data. Subsequently, the differences between the sequenced genomes of male and female individuals were compared to obtain SNP genotyping data, and association analysis with sex was performed to identify SNP variants on the sex chromosome (Z / W chromosome) that are closely linked to the sex trait of the pearl softshell turtle. These variants were named ZZB_SNP, where ZZB is an abbreviation for pearl softshell turtle. Primers were designed to specifically amplify the SNP locus, and population testing was conducted using 16 samples (8 females and 8 males). This further verified that different genotypes of this SNP locus are highly consistent with the sex phenotype of the pearl softshell turtle, and it can be developed into a sex-specific SNP molecular marker for genetic sex identification.

[0036] The genetic sex determination mechanism of the pearl softshell turtle is ZW-type. Females have a ZW sex chromosome composition, while males have a ZZ sex chromosome composition. The ZZB_SNP locus has an A base on the Z chromosome and a G base on the W chromosome. Therefore, in females, the ZZB_SNP locus represents the AG genotype, while in males, it represents the AA genotype.

[0037] Based on this, one embodiment of the present invention provides the application of a sex-specific SNP molecular marker for pearl turtles, or its detection primer pair or detection kit, in the sex identification of pearl turtles; the SNP molecular marker is located at the 128th base of the nucleotide sequence shown in SEQ ID NO. 3 and / or 4, exhibiting A / G polymorphism, and having AG and AA genotypes; wherein, when the 128th base is the AA genotype, the pearl turtle being tested is male, and when the 128th base is the AG genotype, the pearl turtle being tested is female.

[0038] The SNP molecular markers closely linked to the genetic sex traits of pearl softshell turtles provided by this invention offer important molecular tools for sex identification and sex determination mechanism research. By transforming the determination of individual sex traits in pearl softshell turtles into the determination of SNP molecular marker genotypes, sex identification can be performed at various growth stages of pearl softshell turtles without waiting for sexual maturity. This facilitates early targeted breeding in the pearl softshell turtle farming industry, selectively raising faster-growing individuals of the sexes, shortening the all-female breeding time, significantly reducing farming costs, improving farming efficiency and overall yield, and promoting the high-quality and sustainable development of the pearl softshell turtle farming industry. Moreover, this invention performs sex prediction and identification at the genetic level, offering advantages such as speed, efficiency, high throughput, and minimal interference from environmental factors. It greatly improves the screening and identification efficiency and sex selection intensity of pearl softshell turtles, significantly shortens the sex detection cycle, and improves the accuracy and reliability of early sex selection. It has broad application prospects and social significance in the field of pearl softshell turtle sex identification.

[0039] The detection of the SNP molecular marker genotype of this invention can be performed using methods commonly used in the prior art, such as gene chip technology, competitive allele-specific PCR (KASP) technology, Taqman probe technology, high-resolution melting curve (HRM) method, allele-specific PCR (AS-PCR) technology, direct sequencing method, and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry.

[0040] This invention preferably employs a direct sequencing method, including the steps of extracting genomic DNA from the individual softshell turtle to be tested, PCR amplification of the target fragment, and sequencing of the target fragment. In the sequencing peak diagram, a homozygous SNP molecular marker genotype shows a single peak, while a heterozygous genotype shows a double peak, making it easy to distinguish between different genotypes. Specifically, in the sequencing peak diagram, a single peak of A at position 128 indicates the genotype AA, while a double peak of AG indicates the AG genotype.

[0041] Another embodiment of the present invention provides a detection primer pair for detecting the sex-specific SNP molecular marker of the pearl turtle as described above. The detection primer pair includes an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer (F) is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer (R) is shown in SEQ ID NO.2.

[0042] ZZB_SNP_F: AGAAATCAAATCCAATCAG (see SEQ ID NO.1);

[0043] ZZB_SNP_R: TTGATGATTAACTTCTTAC (see SEQ ID NO. 2).

[0044] This invention uses the genomic DNA of the pearl turtle to be tested as a template, performs PCR amplification using the above-mentioned detection primer pair, and sequences the amplified product. It can obtain accurate base information and genotype information of the 128th base of the amplified product, which is the SNP molecular marker site. The primers have high specificity and good molecular marker genotyping effect, which is conducive to the rapid, accurate and efficient identification of SNP molecular marker genotype, and thus the identification of the sex of the pearl turtle individual to be tested.

[0045] In a typical implementation, the nucleotide sequence of the amplification product is shown in SEQ ID NO.3 and / or 4. When the amplification product is detected to include only the nucleotide sequence shown in SEQ ID NO.3, the SNP molecular marker is AA genotype, and the tested pearl turtle individual is predicted or determined to be male; when the amplification product is detected to include both the nucleotide sequences shown in SEQ ID NO.3 and 4, the SNP molecular marker is AG genotype, and the tested pearl turtle individual is predicted or determined to be female.

[0046] In practical applications, since there are other variant sites upstream and downstream of the molecular marker site of the present invention that are not completely linked to sex, the amplification product may also include other sequences besides those shown in SEQ ID NO.3 and / or 4; the variant type (A / G polymorphism) and genotype of the 128th base in these sequences are completely linked to sex. Therefore, in the preferred embodiment of the present invention, the 128th base of the amplification product is used as an independent detection target.

[0047] Another embodiment of the present invention provides a detection kit, the kit comprising the detection primer pair as described above.

[0048] The advantages of the detection kit over the prior art are the same as the advantages of the detection primer pair over the prior art as described above, and will not be repeated here.

[0049] Optionally, the detection kit further includes PCR amplification reagents. The present invention does not have any special limitation on the source of the PCR amplification reagents, and conventional commercially available products in the art can be used.

[0050] In a typical implementation, the PCR amplification reagents include Taq DNA polymerase, dNTPs, and buffer reagents, such as the 2×Taq Master Mix (Dye) kit from Jiangsu Kangwei Century Technology Co., Ltd.

[0051] This invention does not impose specific limitations on the total amount of PCR amplification reagents and detection primers in the kit; the amounts can be set according to the standard requirements of the kit. Generally, the concentrations of the upstream and downstream primers are 10-20 mM, which is usually the concentration of the stock solution.

[0052] Based on the same inventive concept as described above, another embodiment of the present invention provides a method for identifying the sex of a pearl turtle, comprising the following steps:

[0053] Genomic DNA was extracted from the individual pearl softshell turtle to be tested;

[0054] The genomic DNA was amplified by polymerase chain reaction (PCR) using the primer pairs shown in SEQ ID NO.1-2 to obtain the amplification product;

[0055] The genotype of the amplification product at position 128 is detected, and the sex of the individual pearl turtle to be tested is determined based on the genotype.

[0056] Among them, the 128th base of the amplification product has AA and AG genotypes. When it is AA genotype, the tested pearl turtle is male, and when it is AG genotype, the tested pearl turtle is female.

[0057] This invention does not specifically limit the method for extracting genomic DNA from individual pearl softshell turtles to be tested. Commonly used genomic DNA extraction methods in the field can be used, such as the commonly used phenol-chloroform crude extraction method or CTAB extraction method, or commercially available genomic DNA extraction kits, such as the general-purpose column-type genomic DNA extraction kit from Jiangsu Kangwei Century Technology Co., Ltd.

[0058] Optionally, the PCR amplification reaction system, in 40 μL, comprises: 20 μL of 2×Taq Master Mix (Dye), 1.6 μL of upstream primer, 1.6 μL of downstream primer, 1 μL of genomic DNA, and 15.8 μL of deionized water (ddH2O).

[0059] Optionally, the PCR amplification reaction program includes: 94 °C pre-denaturation for 3 min, 94 °C denaturation for 30 s, 50 °C annealing for 30 s, 72 °C extension for 15 s, for a total of 35 cycles; running at 72 °C for 5 min; and storing the amplification reaction solution at 4 °C for later use.

[0060] Optionally, the genotype of the 128th base of the amplification product can be detected by Sanger sequencing.

[0061] The present invention will be further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, such as those described in *Molecular Cloning: A Laboratory Manual (Fourth Edition)* published by Cold Spring Harbor Laboratory, or generally under the conditions recommended by the manufacturer.

[0062] 1. Experimental group

[0063] The 20 pearl softshell turtles used for genome resequencing and the 16 pearl softshell turtles used for population validation of molecular markers were all from the Turtle and Softshell Turtle Breeding and Conservation Research Base of the Pearl River Fisheries Research Institute. After confirming that they were free of infection on their body surface and were active normally, they were used for subsequent genome extraction.

[0064] 2. Gender determination

[0065] The sex phenotype of the tested individuals was determined by collecting sexually mature individuals and observing their gonadal phenotypes after dissection. Pearl softshell turtles were cultured to sexual maturity, and preliminary classification was performed based on morphological observation. Male turtles had long, slender tails that naturally extended beyond the folds of their shells, with a relatively stiff tail tip; female turtles had short, thick tails that did not protrude beyond the folds of their shells, with a softer tail tip. Each individual was then anesthetized in an ice box, and after anesthesia, their entire body was disinfected with 75% ethanol to remove any contaminants. Subsequently, the genital opening was cut open to observe the gonads, ultimately determining their sex.

[0066] 3. Whole-genome resequencing and sex association analysis of the experimental population

[0067] Muscle tissue was collected from three-year-old pearl softshell turtles. Genomic DNA was extracted from the samples using a universal column-type genomic DNA extraction kit (purchased from Jiangsu Kangwei Century Technology Co., Ltd., catalog number CW2298M). The operation steps were performed according to the instruction manual.

[0068] After quality control of the extracted genomic DNA, high-throughput resequencing of the genome was performed using the DNBSEQ platform, with a sequencing depth of approximately 15×, a pair-end 150bp sequencing strategy, and a sequencing throughput of approximately 33Gb. Read alignment and filtering (Clean Reads) were performed on the sequencing data to obtain high-quality whole-genome resequencing data. Base identification was performed on the raw image data obtained from the resequencing using CASAVA software, and these raw sequence data were further converted to FASTQ format. However, these raw data often contain sequencing adapter sequences and low-quality reads; therefore, strict filtering was required before further analysis. SOAPnuke (v2.1.0) software developed by BGI Genomics Co., Ltd. was used to remove reads contaminated by adapters, reads with excessively low base quality (i.e., bases with a quality value of 12 or less accounting for 50%), and reads containing too many N bases (more than 10%). The resulting high-quality Clean Data was used for subsequent analysis (after filtering, Clean Data Q20 was greater than 90%, and Q30 was greater than 80%). The aforementioned work was commissioned to Guangzhou Ruike Gene Technology Co., Ltd.

[0069] The assembled genome was quality controlled, and its mitochondrial sequencing sequence was selected to match the pearl softshell turtle mitochondrial DNA sequence (NCBI Reference Sequence: NC_014054.1, GenBank: KX882744.1) in the NCBI database with a similarity of 99.9%, confirming that the sequencing and assembled data is indeed the genomic data of the pearl softshell turtle, which can be used for the subsequent development of sex-specific molecular markers.

[0070] BWA alignment software was used to align paired-end sequencing data using the MEM alignment strategy. Default alignment parameters were employed. After alignment, samtools (version 1.14) was used to sort the aligned sequences. Following alignment, DeepvaRiant software with default parameters was used to detect SNP variants across the entire genome, and samtoolsmpileup and Python programs were used for secondary verification of molecular marker genotypes.

[0071] Based on whole-genome sequencing data and genome assembly results at the chromosome level, it was confirmed that the pearl softshell turtle has a ZW-type sex determination mechanism. Therefore, SNP loci on the sex chromosomes that meet the criteria of homozygous genotype in males (Z chromosome) and heterozygous genotype in females (Z chromosome and W chromosome) were selected as potential genetic sex-associated molecular markers.

[0072] Based on the gene sequences assembled from high-throughput sequencing data analysis, a sex-specific SNP locus was discovered in male and female samples of pearl softshell turtles, with different genotypes, named ZZB_SNP. ZZB_SNP is located at base position 128 of SEQ ID NO. 3 and / or 4, exhibiting A / G polymorphism (sequence alignment results are shown in...). Figure 4 The sequence shown in SEQ ID NO.3 is located on chromosome Z, and the nucleotide corresponding to ZZB_SNP is A. The sequence shown in SEQ ID NO.4 is located on chromosome W, and the nucleotide corresponding to ZZB_SNP is G. ZZB_SNP has both AG and AA genotypes. In females, this locus has the AG genotype, and in males, it has the AA genotype. The sequence information of ZZB_SNP is as follows, with the underlined and bolded positions indicating the ZZB_SNP sites:

[0073] It should be noted that, in population samples, the variation type of the 128th base (ZZB_SNP) in the sequence shown in SEQ ID NO. 3 and / or 4 is completely linked to the sex phenotype. Although other variation sites exist upstream and downstream of the ZZB_SNP, these variation sites are not completely linked to sex. For example, the A / C polymorphism at position 28 of the sequence shown in SEQ ID NO. 3 and / or 4 may be mutated in some male and female individuals, while being completely identical in others. Therefore, these variation sites are not considered for detection in this invention. Furthermore, based on these variations, sequences containing the ZZB_SNP molecular marker include, but are not limited to, the sequences shown in SEQ ID NO. 3 and / or 4, as well as sequences identical at position 128 of the sequence shown in SEQ ID NO. 3 and / or 4, but with minor variations at other sites.

[0074] 4. Molecular marker validation at the natural population level

[0075] Primers were designed for the aforementioned sex-specific SNP loci. Genomic DNA was extracted from 16 randomly selected population samples (8 females and 8 males) for PCR amplification. Sanger sequencing was then used to verify the SNP genotypes and their association with sex. The final sex of the population samples was confirmed by observing the gonads after dissection. Eight female samples were named ZZB-F1 to ZZB-F8, and eight male samples were named ZZB-M1 to ZZB-M8.

[0076] 4.1 PCR detection primer sequence design

[0077] Based on the genome sequence and upstream and downstream molecular marker sequences assembled from high-throughput sequencing data analysis, PCR validation primer pairs ZZB_SNP_F and ZZB_SNP_R were designed using Primer3 version 4.1.0 software (web link: https: / / bioinfo.ut.ee / primer3-0.4.0 / ). The primer sequences are as follows, with upstream and downstream primers denoted by F and R, respectively:

[0078] ZZB_SNP_F: AGAAATCAAATCCAATCAG (see SEQ ID NO.1);

[0079] ZZB_SNP_R: TTGATGATTAACTTCTTAC (see SEQ ID NO. 2).

[0080] The amplification product is 231 bp in size, and ZZB_SNP is located at the 128th base of the amplification product (nucleotide sequence as shown in SEQ ID NO.3 or 4, with the underlined part indicating the primer sequence).

[0081] 4.2 PCR amplification and band detection

[0082] Muscle tissue was collected from three-year-old pearl softshell turtles, and the genome was extracted using a general-purpose column-type genomic DNA extraction kit from Jiangsu Kangwei Century Technology Co., Ltd.

[0083] PCR amplification was performed using extracted genomic DNA as a template. The PCR amplification system (40 μL) consisted of: 20 μL of 2×TaqMaster Mix (Dye) (purchased from Jiangsu Kangwei Century Technology Co., Ltd., catalog number CW0682L), 1.6 μL of upstream primer, 1.6 μL of downstream primer, 1 μL of genomic DNA, and 15.8 μL of deionized water (ddH2O). The reaction program included: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 50℃ annealing for 30 s, and 72℃ extension for 15 s, for a total of 35 cycles; followed by a 5 min run at 72℃. The amplified reaction solution was stored at 4℃ for later use.

[0084] 3 μL of the amplification product was subjected to electrophoresis on a 1.1% agarose gel at 160V for 30 min. The electrophoresis gel image is shown below. Figure 1As shown, lane M is the molecular marker (DM2000, purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd., catalog number CW0632M). The DM2000 DNA marker consists of six DNA fragments, with lengths of 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp, respectively. Lanes 1-16 represent female samples ZZB-F1 to ZZB-F8 and male samples ZZB-M1 to ZZB-M8, respectively. The amplified fragment size is 231 bp, exhibiting a single target band, demonstrating the good specificity of the detection primers.

[0085] The gel electrophoresis bands were cut off, the amplification products were recovered, and Sanger sequencing was performed. The sequencing peak diagrams of female and male individuals for ZZB_SNP are shown in Figures 2-3. The sequencing results were analyzed using Codoncode software. After removing low-quality bases, the sequences were spliced ​​to obtain the PCR product sequences. Sequence alignment confirmed the genotypes of the SNP loci. The sequence alignment results for female and male individuals are shown in Figures 2-3. Figure 4 .

[0086] In male individuals (sex chromosome ZZ), ZZB_SNP sequencing only yielded individuals with the AA genotype, whose sequencing sequence is shown in SEQ ID NO.3 or is a sequence identical to the sequence shown in SEQ ID NO.3-4 at position 128, with minor variations at other sites; in female individuals (sex chromosome ZW), ZZB_SNP sequencing only yielded individuals with the AG genotype, whose sequencing sequence is shown in SEQ ID NO.3-4 or is a sequence identical to the sequence shown in SEQ ID NO.3-4 at position 128, with minor variations at other sites.

[0087] Sex was classified based on the ZZB_SNP molecular marker genotype: females were heterozygous and males were homozygous, specifically: the genotype of female samples at the ZZB_SNP was AG, and the genotype of male samples was AA. The sex information based on the SNP locus genotype was consistent with the sex information confirmed by physiological anatomy (see Table 1), demonstrating that the ZZB_SNP molecular marker of this invention is completely linked to sex, and its genotype can be used for rapid identification of the genetic sex of pearl softshell turtles, with a sex differentiation accuracy of 100%.

[0088] Table 1. Statistical results of SNP loci genotypes and genetic sex in pearl softshell turtles.

[0089] Serial Number Sample Name Gender (confirmed after autopsy) ZZB_SNP genotype (A / G) ZZB_SNP position 1 ZZB-F1 female AG 128 bp 2 ZZB-F2 female AG 128 bp 3 ZZB-F3 female AG 128 bp 4 ZZB-F4 female AG 128 bp 5 ZZB-F5 female AG 128 bp 6 ZZB-F6 female AG 128 bp 7 ZZB-F7 female AG 128 bp 8 ZZB-F8 female AG 128 bp 9 ZZB-M1 male AA 128 bp 10 ZZB-M2 male AA 128 bp 11 ZZB-M3 male AA 128 bp 12 ZZB-M4 male AA 128 bp 13 ZZB-M5 male AA 128 bp 14 ZZB-M6 male AA 128 bp 15 ZZB-M7 male AA 128 bp 16 ZZB-M8 male AA 128 bp

[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a sex-specific SNP molecular marker for pearl turtles, or its detection primer pair or detection kit, in the sex identification of pearl turtles, characterized in that, The SNP molecular marker is located at the 128th base of the nucleotide sequence shown in SEQ ID NO.3 and 4, exhibiting A / G polymorphism and having AG and AA genotypes; Specifically, when the 128th base is of the AA genotype, the tested pearl turtle is male; when the 128th base is of the AG genotype, the tested pearl turtle is female.

2. The application of the sex-specific SNP molecular marker for pearl turtles, or its detection primer pairs or detection kit, according to claim 1, in the sex identification of pearl turtles, characterized in that, The detection primer pair is used to detect sex-specific SNP molecular markers of pearl turtles. The detection primer pair includes an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

2.

3. The application of the sex-specific SNP molecular marker for pearl turtles, or its detection primer pair or detection kit, according to claim 1, in the sex identification of pearl turtles, characterized in that... The detection kit includes the detection primer pair, which includes an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

2.

4. The application of the sex-specific SNP molecular marker for pearl turtles, or its detection primer pairs or detection kit, according to claim 3, in the sex identification of pearl turtles, characterized in that, The detection kit also includes PCR amplification reagents, which include Taq DNA polymerase, dNTPs, and buffer reagents.

5. A method for determining the sex of a pearl turtle, characterized in that, Includes the following steps: Genomic DNA was extracted from the individual pearl softshell turtle to be tested; The genomic DNA was amplified by polymerase chain reaction using primer pairs as shown in SEQ ID NO.1-2 to obtain amplification products; The genotype of the amplified product at position 128 is detected, and the sex of the individual pearl turtle to be tested is determined based on the genotype. The amplified product has AA and AG genotypes at position 128. When it is AA genotype, the individual to be tested is determined to be male, and when it is AG genotype, the individual to be tested is determined to be female.

6. The method for determining the sex of pearl turtles according to claim 5, characterized in that, If the amplification product is detected to contain only the nucleotide sequence shown in SEQ ID NO.3, the individual turtle being tested is determined to be male; if the amplification product is detected to contain both the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4, the individual turtle being tested is determined to be female.

7. The method for determining the sex of pearl turtles according to claim 5, characterized in that, The polymerase chain reaction amplification reaction system, in 40 μL volume, includes: 20 μL of 2×Taq Master Mix, 1.6 μL of upstream primer, 1.6 μL of downstream primer, 1 μL of genomic DNA, and 15.8 μL of deionized water; The polymerase chain reaction amplification program includes: 94 °C pre-denaturation for 3 min, 94 °C denaturation for 30 s, 50 °C annealing for 30 s, 72 °C extension for 15 s, for a total of 35 cycles; and 72 °C run for 5 min.

8. The method for determining the sex of pearl turtles according to claim 5, characterized in that, The genotype was detected using Sanger sequencing.

Images