Specific snp molecular marker for rapid identification of genetic sex of pelodiscus ferro and application thereof

Abstract

The application belongs to the technical field of molecular marker assisted breeding, and particularly relates to a specific SNP molecular marker for rapidly identifying the genetic sex of Pelochelys cantonensis and application thereof, the SNP molecular marker is selected from at least one of JB_SNP1-3, is located at the 337th, 363rd and / or 392th base of the nucleotide sequence shown in SEQ ID NO. 3 and / or 4, and when being GG, CC and / or TT genotype respectively, the to-be-tested Pelochelys cantonensis individual is male, and when being GT, CA and / or TC genotype respectively, the to-be-tested Pelochelys cantonensis individual is female. The application provides an important molecular tool for the sex identification and sex determination mechanism research of Pelochelys cantonensis, can convert the judgment of the sex of Pelochelys cantonensis into the judgment of the SNP genotype, is favorable for the sex identification of Pelochelys cantonensis in the early growth stage, and has the advantages of rapidness and high efficiency, short sex detection period, large detection flux, small environmental factor interference, high accuracy and reliability of detection results and the like.

Description

Technical Field

[0001] This invention relates to the field of molecular marker-assisted breeding technology, and in particular to a specific SNP molecular marker for rapid identification of the genetic sex of the horned turtle and its application. Background Technology

[0002] The spiny softshell turtle (Apalone spinifera), belonging to the family Trionychidae, subfamily Trionycisinae, and genus Apalone, is native to central North America and has been introduced to my country in recent years. It is a relatively large turtle, with adults reaching 45 cm in length and weighing 3-5 kg, classifying it as a medium-to-large-sized turtle. Adults have a flat, oval body shape with a wide, thin skirt. The carapace is grayish-green with large ring-shaped spots, and a black line or dark marking along the edge, giving it high ornamental value. Furthermore, the meat of the spiny softshell turtle is delicious and nutritious, possessing properties that clear heat and nourish yin, calm the liver and extinguish wind, and soften and disperse lumps. It not only has high edible value but is also a widely used tonic and traditional Chinese medicine ingredient. Therefore, whether farmed or kept as an ornamental turtle, the spiny softshell turtle is a freshwater animal with extremely high economic value and good market development potential.

[0003] The economic value of horned softshell turtles differs significantly between sexes. Females of the same age are much larger than males, exhibiting obvious sexual dimorphism. Furthermore, under natural reproduction conditions, males are fewer and smaller, while females are more numerous and larger. During mating, females often compete for and injure males, leading to low fertilization and hatching rates. Therefore, in large-scale horned softshell turtle farming, sex determination is often necessary early in development to achieve monosex farming and efficient all-female breeding for commercial purposes, significantly improving the efficiency and profitability of the farming industry while reducing costs.

[0004] Currently, there is very little literature on horned 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] By utilizing molecular markers closely linked to genetic sex, and through DNA extraction and PCR detection, molecular marker sites in candidate individuals can be identified and selected. 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 has not yet been conducted on the horned softshell turtle (Trionyx sinensis), and online databases such as NCBI lack its complete genome sequence, making the development of molecular sex determination technology for this species particularly difficult. Therefore, exploring the sex determination mechanism of the horned 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 horned softshell turtle, and is of great significance for reducing the cost of horned softshell turtle farming and improving its economic benefits. Summary of the Invention

[0006] To address the lack of sex-specific molecular markers for horned 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 horned turtles. This provides an important molecular tool for sex identification and sex determination mechanism research in horned 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 horned turtle sex identification. This invention is specifically implemented through the following technical solutions:

[0007] The first aspect of this invention provides the application of a sex-specific SNP molecular marker for horned turtles, or its detection primer pair or detection kit, in sex identification of horned turtles; the SNP molecular marker is selected from at least one of JB_SNP1 to JB_SNP3, and JB_SNP1 to JB_SNP3 are respectively located at the 337th, 363rd, and / or 392nd bases of the nucleotide sequence shown in SEQ ID NO. 3 and / or 4, wherein:

[0008] The 337th base exhibits a G / T polymorphism, with GG and GT genotypes. When the 337th base is the GG genotype, the tested horned turtle is male, and when it is the GT genotype, the tested horned turtle is female.

[0009] The 363rd base exhibits a C / A polymorphism, with CC and CA genotypes. When the 363rd base is the CC genotype, the individual tested is male; when it is the CA genotype, the individual tested is female.

[0010] The 392nd base exhibits a T / C polymorphism, possessing both TT and TC genotypes. When the 392nd base is of the TT genotype, the tested horned turtle individual is male; when it is of the TC genotype, the tested horned turtle individual is female.

[0011] A second aspect of the present invention provides a detection primer pair for detecting sex-specific SNP molecular markers of horned turtles, wherein the SNP molecular markers are located at positions 337, 363, and / or 392 of the nucleotide sequence shown in SEQ ID NO. 3 and / or 4.

[0012] 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.

[0013] A third aspect of the present invention provides a detection kit comprising the detection primer pairs described above.

[0014] Furthermore, the detection kit also includes PCR amplification reagents, which include Taq DNA polymerase, dNTPs, and buffer reagents.

[0015] The fourth aspect of this invention provides a method for identifying the sex of a horned turtle, comprising the following steps:

[0016] Genomic DNA was extracted from the individual softshell turtle to be tested;

[0017] The genomic DNA was amplified by polymerase chain reaction using primer pairs as shown in SEQ ID NO.1-2 to obtain amplification products;

[0018] The genotype of the amplified product at positions 337, 363, and / or 392 is detected, and the sex of the individual softshell turtle to be tested is determined based on the genotype; wherein:

[0019] The amplified product exhibits a G / T polymorphism at the 337th base, with GG and GT genotypes. When the 337th base is the GG genotype, the tested horned turtle is male, and when it is the GT genotype, the tested horned turtle is female.

[0020] The amplified product exhibits a C / A polymorphism at the 363rd base, with CC and CA genotypes. When the 363rd base is of the CC genotype, the tested horned turtle is male, and when it is of the CA genotype, the tested horned turtle is female.

[0021] The amplified product exhibits a T / C polymorphism at position 392, with TT and TC genotypes. When position 392 is the TT genotype, the tested horned turtle is male, and when it is the TC genotype, the tested horned turtle is female.

[0022] Furthermore, if the amplification product is detected to include only the nucleotide sequence shown in SEQ ID NO.3, the individual turtle 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 turtle to be tested is determined to be female.

[0023] 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.

[0024] Furthermore, the PCR amplification reaction program includes: 94 °C pre-denaturation for 3 min, 94 °C denaturation for 30 s, 52 °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.

[0025] Furthermore, the genotype was detected using Sanger sequencing.

[0026] The advantages and positive effects of this invention are as follows:

[0027] The SNP molecular markers closely linked to the genetic sex traits of horned turtles provided by this invention offer important molecular tools for sex identification and sex determination mechanism research. They can transform the determination of individual sex traits in horned turtles into the determination of SNP molecular marker genotypes, enabling sex identification at all growth stages of horned turtles. This is beneficial for early targeted breeding in the horned turtle farming industry and has advantages such as speed and efficiency, short sex detection cycle, high detection throughput, minimal interference from environmental factors, high accuracy and reliability of detection results. It has broad application prospects and social significance in the field of horned turtle sex identification, and is of great significance for improving farming efficiency and overall yield, and promoting the high-quality and sustainable development of horned turtle farming. Attached Figure Description

[0028] 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.

[0029] Figure 1This is an agarose gel electrophoresis image of the sex-specific SNP molecular marker amplification products of the horned turtle according to an embodiment of the present invention;

[0030] Figure 2 This is a sequencing peak diagram of the amplified product of the sex-specific SNP molecular marker JB_SNP1 of the horned turtle in an embodiment of the present invention;

[0031] Figure 3 This is a sequencing peak diagram of the amplified product of the sex-specific SNP molecular marker JB_SNP2 of the horned turtle in an embodiment of the present invention;

[0032] Figure 4 This is a sequencing peak diagram of the amplified product of the sex-specific SNP molecular marker JB_SNP3 of the horned turtle in an embodiment of the present invention;

[0033] Figure 5 This is a sequencing comparison diagram of different genotypes of the sex-specific SNP molecular marker JB_SNP1-3 in male and female individuals of the horned turtle according to an embodiment of the present invention.

[0034] Figure 6 This is a diagram showing the comparison results between the mitochondrial DNA of the horned turtle and the NCBI online database in an embodiment of the present invention. Detailed Implementation

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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 sex in horned turtles will provide important molecular tools for in-depth research on the sex determination mechanisms of horned turtles and other turtle species, offering effective means of early sex identification in horned turtles and providing strong support for the sustainable development of horned turtle aquaculture.

[0041] Since the horned softshell turtle lacks complete whole-genome information, this invention first performed whole-genome resequencing on 20 randomly selected horned softshell turtles. The raw high-throughput sequencing data was then quality-filtered and assembled to obtain the genome 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. Three SNP variant sites closely linked to the sex trait of the horned softshell turtle on the sex chromosome (Z / W chromosome) were identified and named JB_SNP1-3, where JB is the abbreviation for horned softshell turtle. Primers were designed to specifically amplify the SNP sites, and population testing was conducted using 16 samples (8 females and 8 males). This further verified that the different genotypes of the aforementioned SNP sites were highly consistent with the sex phenotype of the horned softshell turtle, and can be developed into sex-specific SNP molecular markers for genetic sex identification.

[0042] The genetic sex determination mechanism of the horned softshell turtle is ZW-type. Females have a ZW sex chromosome composition, while males have a ZZ sex chromosome composition. Specifically, the JB_SNP1 locus has a G base on the Z chromosome and a T base on the W chromosome, possessing both GG and GT genotypes. In females, this locus has the GT genotype, while in males, it has the GG genotype. JB_SNP2 has a C base on the Z chromosome and an A base on the W chromosome, possessing both CC and CA genotypes. In females, this locus has the CA genotype, while in males, it has the CC genotype. JB_SNP3 has a T base on the Z chromosome and a C base on the W chromosome, possessing both TT and TC genotypes. In females, this locus has the TC genotype, while in males, it has the TT genotype. Furthermore, the GT+CA+TC genotype combinations of JB_SNP1, JB_SNP2, and JB_SNP2 are completely linked in female individuals, while the GG+CC+TT genotype combination is completely linked in male individuals. Any one of these three genotypes or their combination can be used for sex identification.

[0043] Based on this, one embodiment of the present invention provides the application of a sex-specific SNP molecular marker for horned turtles, or its detection primer pair or detection kit, in the sex identification of horned turtles; the SNP molecular marker is selected from at least one of JB_SNP1 to JB_SNP3, and JB_SNP1 to JB_SNP3 are respectively located at the 337th, 363rd, and / or 392nd bases of the nucleotide sequence shown in SEQ ID NO. 3 and / or 4; wherein:

[0044] The 337th base exhibits a G / T polymorphism, with GG and GT genotypes. When the 337th base is GG, the tested horned turtle is male; when it is GT, the tested horned turtle is female. The 363rd base exhibits a C / A polymorphism, with CC and CA genotypes. When the 363rd base is CC, the tested horned turtle is male; when it is CA, the tested horned turtle is female. The 392nd base exhibits a T / C polymorphism, with TT and TC genotypes. When the 392nd base is TT, the tested horned turtle is male; when it is TC, the tested horned turtle is female.

[0045] The SNP molecular markers closely linked to the genetic sex traits of horned 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 horned turtles into the determination of SNP molecular marker genotypes, sex identification can be performed at various growth stages of horned turtles without waiting for sexual maturity. This facilitates early targeted breeding in the horned turtle farming industry, selectively raising faster-growing individuals of the sexes and 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 horned turtle farming. 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 horned turtle males and females, 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 horned turtle sex identification.

[0046] 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.

[0047] 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.

[0048] Another embodiment of the present invention provides a detection primer pair for detecting the sex-specific SNP molecular marker of the horned 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.

[0049] JB_SNP_F: TAGACAGGAGCTAGATTAC (see SEQ ID NO.1);

[0050] JB_SNP_R: ATAAATCAGCAGAAAATCA (see SEQ ID NO. 2).

[0051] This invention uses the genomic DNA of the horned turtle to be tested as a template, performs PCR amplification using the above-mentioned detection primer pairs, and sequences the amplification products to obtain accurate base information of SNP molecular marker sites. 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 genotypes, and thus the identification of the sex of the horned turtle individual to be tested.

[0052] In a typical implementation, the nucleotide sequence of the amplification product is as shown in SEQ ID NO.3 and / or 4, with JB_SNP1-3 located at bases 337, 363, and / or 392 of the aforementioned nucleotide sequence, respectively. When the amplification product is detected to include only the nucleotide sequence shown in SEQ ID NO.3, and JB_SNP1-3 represents the GG, CC, and TT genotypes, the tested horned 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, and JB_SNP1-3 represents the GT, CA, and TC genotypes, the tested horned turtle individual is predicted or determined to be female.

[0053] In practical applications, since there are other variant sites upstream of the molecular marker site JB_SNP1-3 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 types and genotypes of the bases at positions 337, 363, and 392 in these sequences are completely linked to sex. Therefore, in a preferred embodiment of the present invention, the bases at positions 337, 363, and / or 392 of the amplification product are used as independent detection targets.

[0054] Another embodiment of the present invention provides a detection kit, the kit comprising the detection primer pair as described above.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] Based on the same inventive concept as described above, another embodiment of the present invention provides a method for identifying the sex of a horned turtle, comprising the following steps:

[0060] Genomic DNA was extracted from the individual softshell turtle to be tested;

[0061] 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;

[0062] The genotype of the amplified product at positions 337, 363, and / or 392 is detected, and the sex of the individual softshell turtle to be tested is determined based on the genotype; wherein:

[0063] The amplification product exhibits a G / T polymorphism at base position 337, with both GG and GT genotypes. When the base position 337 is GG, the tested horned turtle is male; when it is GT, the tested horned turtle is female. The amplification product exhibits a C / A polymorphism at base position 363, with both CC and CA genotypes. When the base position 363 is CC, the tested horned turtle is male; when it is CA, the tested horned turtle is female. The amplification product exhibits a T / C polymorphism at base position 392, with both TT and TC genotypes. When the base position 392 is TT, the tested horned turtle is male; when it is TC, the tested horned turtle is female.

[0064] This invention does not specifically limit the method for extracting genomic DNA from individual softshell turtles. 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.

[0065] 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).

[0066] Optionally, the PCR amplification reaction program includes: 35 cycles of pre-denaturation at 94 °C for 3 min, denaturation at 94 °C for 30 s, annealing at 52 °C for 30 s, extension at 72 °C for 15 s; running at 72 °C for 5 min; and storing the amplification reaction solution at 4 °C for later use.

[0067] Optionally, the genotype of the 128th base of the amplification product can be detected by Sanger sequencing.

[0068] 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.

[0069] 1. Experimental group

[0070] The 20 horned turtles used for genome resequencing and the 16 horned turtles used for population validation of molecular markers were all from the Turtle and Soft-shelled 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.

[0071] 2. Gender determination

[0072] The sex phenotype of the tested individuals was determined by collecting sexually mature individuals and observing their gonadal phenotypes after dissection. The horned turtles were cultured to sexual maturity, and preliminary classification was performed based on morphological observation. Among individuals of the same age, females were larger, while males were much smaller; males had thinner and longer bodies, narrower limbs, and tails that clearly extended beyond the genital folds; females had rounder and thicker bodies, wider limbs, and tails that did not extend beyond the genital folds. Each individual was then anesthetized in an ice box. After anesthesia, the entire body was disinfected with 75% ethanol to remove contaminants, and then the genital opening was cut open to observe the gonads, ultimately determining the sex.

[0073] 3. Whole-genome resequencing and sex association analysis of the experimental population

[0074] Muscle tissue was collected from three-year-old horned 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.

[0075] 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.

[0076] The assembled genome underwent quality control. The mitochondrial sequencing sequence (nucleotide sequence consisting of SEQ ID NO. 5+6) was selected and found to have a similarity of over 99% with the mitochondrial DNA sequence of the horned turtle (Apalone spinifera mitochondrion NCBIReference Sequence: NC_021371.1, GenBank: KX882745.1) in the NCBI database. The sequence alignment results are shown below. Figure 6 The sequencing and assembly data were confirmed to be genomic data of the horned turtle, which can be used for the subsequent development of sex-specific molecular markers.

[0077] Using BWA alignment software, the MEM alignment strategy was employed to align paired-end sequencing data with default alignment parameters. After alignment, the aligned sequences were sorted using samtools (version 1.14). Following alignment, DeepvaRiant software with default parameters was used to detect SNP variants across the entire genome, and samtoolsmpileup and Python were used for secondary verification of molecular marker genotypes. The horned turtle exhibits a ZW-type sex determination mechanism. SNP loci on the sex chromosomes that conform to the homozygous genotype in males (Z chromosome) and the heterozygous genotype in females (both Z and W chromosomes) were selected as potential genetic sex-associated molecular markers.

[0078] Based on the gene sequences assembled from high-throughput sequencing data analysis, three sex-specific SNP sites with different genotypes were identified in male and female samples of horned turtles, named JB_SNP1-3. JB_SNP1-3 are located at bases 337, 363, and 392 of the nucleotide sequences shown in SEQ ID NO. 3 and / or 4, respectively, and exhibit G / T, C / A, and T / C polymorphisms, respectively (sequence alignment results are shown in...). Figure 4 Of these, the sequence shown in SEQ ID NO.3 is located on chromosome Z, and the sequence shown in SEQ ID NO.4 is located on chromosome W.

[0079] JB_SNP1 (base at position 337) corresponds to G and T nucleotides in SEQ ID NO.3-4, and has GG and GT genotypes. In female individuals, this site has the GT genotype, and in male individuals, this site has the GG genotype.

[0080] JB_SNP2 (base at position 363) corresponds to nucleotides C and A in SEQ ID NO.3-4, and has genotypes CC and CA. In female individuals, this site has the CA genotype, while in male individuals, this site has the CC genotype.

[0081] JB_SNP3 (base at position 392) corresponds to nucleotides T and C in SEQ ID NO.3-4, and has genotypes TT and TC. In female individuals, this site has the TC genotype, and in male individuals, this site has the TT genotype.

[0082] The sequence information for JB_SNP1-3 is as follows, with the underlined and bolded positions indicating the JB_SNP1-3 sites from front to back:

[0083]

[0084]

[0085] It should be noted that, in the analysis and verification of samples from a population of horned turtles, the variant types of JB_SNP1-3 in the sequences shown in SEQ ID NO.3 and / or 4 are completely linked to the sex phenotype. Although other variant sites exist upstream of JB_SNP1, these variant sites are not completely linked to sex. For example, the C / A polymorphism at position 23 may be mutated in some male and female individuals, while remaining completely identical in others. Therefore, these variant sites are not considered for detection in this invention. Furthermore, based on these variants, sequences containing the JB_SNP1-3 molecular marker include, but are not limited to, the sequences shown in SEQ ID NO.3 and / or 4, as well as sequences identical to those shown in SEQ ID NO.3 and / or 4 at positions 337, 363, and 392, but with minor variations at other sites.

[0086] 4. Molecular marker validation at the natural population level

[0087] 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 JB-F1 to JB-F8, and eight male samples were named JB-M1 to JB-M8.

[0088] 4.1 PCR detection primer sequence design

[0089] Based on the genome sequence and upstream and downstream sequences of molecular markers assembled from high-throughput sequencing data analysis, PCR validation primer pairs JB_SNP_F and JB_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:

[0090] JB_SNP_F: TAGACAGGAGCTAGATTAC (see SEQ ID NO.1);

[0091] JB_SNP_R: ATAAATCAGCAGAAAATCA (see SEQ ID NO. 2).

[0092] The amplification product is 412 bp in size, and the nucleotide sequence is shown in SEQ ID NO.3 or 4. The underlined part indicates the primer sequence; JB_SNP1-3 are located at bases 337, 363 and 392 of the amplification product, respectively.

[0093] 4.2 PCR amplification and band detection

[0094] Muscle tissue was collected from three-year-old horned softshell turtles, and the genome was extracted using a general-purpose column-type genomic DNA extraction kit from Jiangsu Kangwei Century Technology Co., Ltd.

[0095] 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, 52℃ 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.

[0096] 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 1 As 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 JB-F1 to JB-F8 and male samples JB-M1 to JB-M8, respectively. The amplified fragment size is 412 bp, exhibiting a single target band, demonstrating the good specificity of the detection primers.

[0097] The gel electrophoresis bands were cut off, the amplification products were recovered, and Sanger sequencing was performed. The sequencing peak diagrams for female and male individuals of JB_SNP1-3 are shown in Figures 2-4, respectively. 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 Figure 2-4. Figure 5 .

[0098] In male individuals (sex chromosome ZZ), JB_SNP1-3 sequencing only yielded individuals with the GG, CC, and TT genotypes. Their sequencing sequences are shown in SEQ ID NO.3 or are sequences identical to the sequence shown in SEQ ID NO.3 at positions 337, 363, and 392, with minor variations at other sites. In female individuals (sex chromosome ZW), JB_SNP1-3 sequencing only yielded individuals with the GT, CA, and TC genotypes. Their sequencing sequences are shown in SEQ ID NO.3-4 or are sequences identical to the sequence shown in SEQ ID NO.3-4 at positions 337, 363, and 392, with minor variations at other sites.

[0099] Sex was classified based on the JB_SNP molecular marker genotype. Females were heterozygous, and males were homozygous, as shown by the following genotypes at JB_SNP1-3 (bases 337, 363, and 392): GT, CA, and TC in female samples, and GG, CC, and TT in males. Furthermore, the GT+CA+TC genotype combination of JB_SNP1, JB_SNP2, and JB_SNP2 was completely linked in females, and the GG+CC+TT genotype combination was completely linked in males. Any one of these three genotypes, or their combination, could be used for sex identification. The sex information obtained from the aforementioned SNP sites or their combinations was consistent with the sex information confirmed by physiological anatomy (see Table 1), demonstrating that the molecular markers of this invention are completely linked to sex, and their genotypes can be used for rapid identification of the genetic sex of horned turtles, achieving a 100% accuracy rate in distinguishing between males and females.

[0100] Table 1. Statistical results of SNP loci genotypes and genetic sex of horned turtles.

[0101]

[0102] 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 detection primer pair or detection kit for sex-specific SNP molecular markers of horned turtles in sex identification of horned turtles, characterized in that, The SNP molecular marker is selected from at least one of JB_SNP1 to JB_SNP3, and JB_SNP1 to JB_SNP3 are located at positions 337, 363, and 392 of the nucleotide sequence shown in SEQ ID NO. 3 and / or 4, respectively. The 337th base exhibits a G / T polymorphism, with GG and GT genotypes. When the 337th base is the GG genotype, the tested horned turtle is male, and when it is the GT genotype, the tested horned turtle is female. The 363rd base exhibits a C / A polymorphism, with CC and CA genotypes. When the 363rd base is of the CC genotype, the individual tested is male; when it is of the CA genotype, the individual tested is female. The 392nd base exhibits a T / C polymorphism, possessing both TT and TC genotypes. When the 392nd base is of the TT genotype, the tested horned turtle individual is male; when it is of the TC genotype, the tested horned turtle individual is female.

2. The application of the detection primer pair or detection kit for sex-specific SNP molecular markers of the horned turtle according to claim 1 in the sex identification of the horned turtle, characterized in that, The detection primer pair is used to detect sex-specific SNP molecular markers of the horned turtle. 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 detection primer pair or detection kit for sex-specific SNP molecular markers of the horned turtle according to claim 1 in the sex identification of the horned turtle, 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 detection primer pair or detection kit for sex-specific SNP molecular markers of the horned turtle according to claim 3 in the sex identification of the horned turtle, characterized in that, The detection kit also includes polymerase chain reaction (PCR) amplification reagents, which include Taq DNA polymerase, dNTPs, and buffer reagents.

5. A detection primer pair, characterized in that, The detection primer pair is used to detect sex-specific SNP molecular markers of the horned turtle, wherein the SNP molecular markers are located at positions 337, 363, and 392 as shown in SEQ ID NO.3 and / or 4; 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.

6. A test kit, characterized in that, The detection kit includes the detection primer pair as described in claim 5.

7. A method for determining the sex of a horned turtle, characterized in that, Includes the following steps: Genomic DNA was extracted from the individual 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 positions 337, 363, and 392 is detected, and the sex of the tested softshell turtle is determined based on the genotype; wherein: The amplified product exhibits a G / T polymorphism at the 337th base, with GG and GT genotypes. When the 337th base is the GG genotype, the tested horned turtle is male, and when it is the GT genotype, the tested horned turtle is female. The amplified product exhibits a C / A polymorphism at the 363rd base, with CC and CA genotypes. When the 363rd base is of the CC genotype, the tested horned turtle is male, and when it is of the CA genotype, the tested horned turtle is female. The amplified product exhibits a T / C polymorphism at position 392, with TT and TC genotypes. When position 392 is the TT genotype, the tested horned turtle is male, and when it is the TC genotype, the tested horned turtle is female.

8. The method for identifying the sex of a horned turtle according to claim 7, 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.

9. The method for determining the sex of a horned turtle according to claim 7, 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 procedure includes: pre-denaturation at 94 °C for 3 min; denaturation at 94 °C for 30 s, annealing at 52 °C for 30 s, extension at 72 °C for 15 s, for a total of 35 cycles; and running at 72 °C for 5 min.

10. The method for identifying the sex of a horned turtle according to claim 7, characterized in that, The genotype was detected using Sanger sequencing.

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