A snp marker related to genetic sex of chinese soft-shelled turtle and primer and application thereof

Abstract

The application discloses a Chinese soft-shelled turtle genetic sex related SNP marker, a primer thereof and application, and relates to the field of genetic sex identification of Chinese soft-shelled turtles. The Chinese soft-shelled turtle genetic sex related SNP marker is a sequence shown in SEQ ID NO:1, wherein bases at positions 164, 167, 172, 173, 174 and 177 from the 5' end are A / G, C / T, T / C, T / C, G / A and C / T. An individual with the SNP marker genotype AG, CT, TC, TC, GA and CT is a female Chinese soft-shelled turtle, and an individual with the SNP marker genotype AA, CC, TT, TT, GG and CC is a male Chinese soft-shelled turtle. The application can quickly, simply and accurately identify the genetic sex of Chinese soft-shelled turtles through corresponding specific marker primers. The method for detecting the genetic sex of Chinese soft-shelled turtles can be used for sex identification and screening of Chinese soft-shelled turtles in different growth stages, different groups and a large number of samples, and has important significance for carrying out sex control breeding of Chinese soft-shelled turtles and developing single-sex breeding.

Description

Technical Field

[0001] This invention relates to the field of SNP technology, and in particular to a genetic sex-related SNP marker for the Chinese soft-shelled turtle, its primers, and applications. Background Technology

[0002] The Chinese softshell turtle (Pelodiscus sinensis), also known as the turtle, is a species of softshell turtle belonging to the phylum Vertebrata, class Reptilia, order Testudines, family Trionychidae, and genus Pelodiscus. It is an amphibious reptile that breathes with lungs and is mainly distributed in China, Japan, Korea, Russia, and Vietnam. The Chinese softshell turtle is high in protein, providing many essential amino acids, and has extremely high nutritional value, making it widely popular among consumers.

[0003] Significant growth differences exist between male and female Chinese soft-shelled turtles. The sex determination mechanism of these turtles remains unclear, making the exploration of sex control technologies and the achievement of male asexual reproduction a crucial direction for Chinese soft-shelled turtle breeding. In the farming process, males generally grow faster than females, with the difference being particularly pronounced at maturity. Males grow 25-30% faster than females, and their thicker skirts and less fat contribute to their higher nutritional value and market price. Therefore, the implementation of asexual breeding of Chinese soft-shelled turtle seedlings has significant economic implications for the development of the Chinese soft-shelled turtle industry. However, before gonadal maturity, sex is difficult to determine visually, with almost no morphological difference between males and females during the embryonic and juvenile stages. This presents significant challenges to the development of asexual breeding technologies and hinders research into the molecular mechanisms of sex determination, which require genetic sex identification before gonadal differentiation.

[0004] Therefore, developing a molecular marker that can accurately identify the genetic sex of Chinese soft-shelled turtles for sex control breeding has great value for production applications and will also promote research on the molecular mechanisms of genome selection breeding and sex determination in Chinese soft-shelled turtles. Summary of the Invention

[0005] 1. Technical problems to be solved

[0006] The purpose of this invention is to solve the problem in the prior art that it is difficult to determine the sex of Chinese soft-shelled turtles by the naked eye before the gonads mature, and that there is almost no difference in morphology between male and female fish in the embryonic and juvenile stages. The invention proposes a genetic sex-related SNP marker for Chinese soft-shelled turtles, its primers, and its application.

[0007] 2. Technical Solution

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A genetic sex-related SNP marker for the Chinese soft-shelled turtle, wherein the SNP marker is the sequence shown in SEQ ID NO:1, with bases A / G, C / T, T / C, T / C, G / A, and C / T at positions 164, 167, 172, 173, 174, and 177 from the 5' end.

[0010] Preferably, individuals with SNPs genotypes of AG, CT, TC, TC, GA, and CT are female Chinese softshell turtles, and individuals with SNPs genotypes of AA, CC, TT, TT, GG, and CC are male Chinese softshell turtles.

[0011] Preferably, the nucleotide sequence of the primer set is shown in SEQ ID NO:2-4.

[0012] This invention also proposes a primer set for genetic sex-related SNPs markers of the Chinese soft-shelled turtle, containing primer sets with nucleotide sequences as shown in SEQ ID NO:2-4.

[0013] This invention also proposes a kit for genetic sex-related SNP markers of the Chinese soft-shelled turtle, containing primer sets with nucleotide sequences as shown in SEQ ID NO:2-4.

[0014] Preferably, its use in identifying the genetic sex of the Chinese soft-shelled turtle.

[0015] This invention also proposes a method for identifying the genetic sex of Chinese soft-shelled turtles. By detecting SNP markers in the Chinese soft-shelled turtles to be tested, the genetic sex of the turtles to be tested can be determined. Individuals with SNP markers of AG, CT, TC, TC, GA, and CT genotypes are female Chinese soft-shelled turtles, and individuals with SNP markers of AA, CC, TT, TT, GG, and CC genotypes are male Chinese soft-shelled turtles.

[0016] Preferably, a method for identifying the genetic sex of the Chinese soft-shelled turtle includes the following steps:

[0017] Step 1: Extract genomic DNA from the Chinese soft-shelled turtle to be tested;

[0018] Step 2: Using a primer set, the genomic DNA of the Chinese soft-shelled turtle to be tested is amplified by PCR to obtain the PCR amplification product;

[0019] Step 3: Perform electrophoresis on the PCR amplification products to determine the genetic sex of the Chinese soft-shelled turtle based on the number and size of the PCR amplification bands.

[0020] Preferably, when the PCR amplification bands are two bands of 265bp and 401bp respectively, the SNPs are labeled as AG, CT, TC, TC, GA, CT genotypes, and the individual is a genetically female Chinese softshell turtle; when the PCR amplification band is a single band of 401bp, the SNPs are labeled as AA, CC, TT, TT, GG, CC genotypes, and the individual is a genetically male Chinese softshell turtle.

[0021] 3. Beneficial effects

[0022] Compared with the prior art, the advantages of this invention are:

[0023] (1) In this invention, three specific marker primers were designed. After verification in different populations, it was found that the specific marker primer set can quickly, easily and accurately identify the genetic sex of different strains of Chinese soft-shelled turtle. This lays the foundation for the successful development of sex control breeding of Chinese soft-shelled turtle, the development of single-sex farming, and the research on the genome selection breeding and sex determination mechanism of Chinese soft-shelled turtle.

[0024] (2) In this invention, the six SNPs involved are all heterozygous in female individuals and homozygous in male individuals, which can ensure the specific binding of primers and ensure the accuracy of typing. Attached Figure Description

[0025] Figure 1 This is a sequence comparison and primer design diagram illustrating the genomic SNP differences between male and female Chinese soft-shelled turtles proposed in this invention.

[0026] Figure 2a The sequencing peak diagram of the SNP sites shown in the 10 female Chinese softshell turtles in Example 2 (the arrows in the figure indicate the SNP sites).

[0027] Figure 2b The sequencing peak diagram of the SNP sites shown in the diagram of 10 male Chinese softshell turtles in Example 2 (the arrows in the diagram indicate the SNP sites).

[0028] Figure 3 This is an electrophoresis result of the genetic sex determination of the Dongting strain of Chinese soft-shelled turtle in Example 3;

[0029] Figure 4 Genetic sex identification diagrams for the Chinese soft-shelled turtle strains from Taiwan, Huangsha, and Japan, as shown in Example 3. Detailed Implementation

[0030] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0031] Example 1:

[0032] A genetic sex-related SNP marker for the Chinese soft-shelled turtle, wherein the SNP marker is the sequence shown in SEQ ID NO:1, with bases at positions 164, 167, 172, 173, 174, and 177 from the 5' end being A / G, C / T, T / C, T / C, G / A, and C / T.

[0033] In this embodiment, individuals with SNPs labeled as AG, CT, TC, TC, GA, CT genotypes are female Chinese softshell turtles, and individuals with SNPs labeled as AA, CC, TT, TT, GG, CC genotypes are male Chinese softshell turtles.

[0034] In this embodiment, the nucleotide sequence of the primer set is shown in SEQ ID NO:2-4.

[0035] In this embodiment, a primer set for genetic sex-related SNPs markers of the Chinese soft-shelled turtle includes primer sets with nucleotide sequences as shown in SEQ ID NO:2-4.

[0036] In this embodiment, a kit for identifying sex-related SNPs in Chinese soft-shelled turtles is also proposed, comprising primer sets with nucleotide sequences as shown in SEQ ID NO:2-4.

[0037] In this embodiment, a method for identifying the genetic sex of Chinese soft-shelled turtles is provided. The genetic sex of the Chinese soft-shelled turtles to be tested is determined by detecting SNPs markers. Individuals with SNPs markers of AG, CT, TC, TC, GA, and CT genotypes are female Chinese soft-shelled turtles, and individuals with SNPs markers of AA, CC, TT, TT, GG, and CC genotypes are male Chinese soft-shelled turtles.

[0038] In this embodiment, a method for identifying the genetic sex of the Chinese soft-shelled turtle includes the following steps:

[0039] Step 1: Extract genomic DNA from the Chinese soft-shelled turtle to be tested;

[0040] Step 2: Using a primer set, the genomic DNA of the Chinese soft-shelled turtle to be tested was amplified by PCR to obtain the PCR amplification product;

[0041] Step 3: Perform electrophoresis on the PCR amplification products to determine the genetic sex of the Chinese soft-shelled turtle based on the number and size of the PCR amplification bands.

[0042] In this embodiment, if the PCR amplification bands are two bands of 265bp and 401bp respectively, then the SNPs are labeled as AG, CT, TC, TC, GA, CT genotypes, and the individual is a genetically female Chinese softshell turtle; if the PCR amplification band is a single band of 401bp, then the SNPs are labeled as AA, CC, TT, TT, GG, CC genotypes, and the individual is a genetically male Chinese softshell turtle.

[0043] Example 2:

[0044] It has the implementation content of the above embodiments, wherein the specific implementation methods of the above embodiments can be referred to the above description, and the embodiments here will not be described in detail again; however, the difference between the embodiments in this application and the above embodiments is that:

[0045] Discovery and validation of sex-specific SNP markers in the Chinese soft-shelled turtle:

[0046] In this embodiment, it has been reported that the Chinese softshell turtle is a diploid organism with a ZZ / ZW sex determination system, with females being ZW and males ZZ. Comparative analysis of 12 sets of whole-genome resequencing data from male and female Chinese softshell turtles revealed multiple SNPs on the seventh exon of the ZNRF3 gene in Scaffold_JH207701.1. These SNPs were all heterozygous in females and homozygous at the corresponding positions in males, suggesting that other mutants at these SNP sites may originate from the W chromosome of female Chinese softshell turtles.

[0047] In this embodiment, to verify this, eight male and eight female Chinese softshell turtles, which had been dissected and sexed for identification, were used. Primer pairs (14749-F and 14749-R) were designed based on the seventh exon sequence of the ZNRF3 gene for amplification. The PCR products were then subjected to Sanger sequencing to examine the peak patterns. The results showed that all eight female individuals were heterozygous at multiple loci, and some SNPs, such as... Figure 2a As shown; all 8 male individuals were homozygous at the corresponding positions, such as Figure 2b As shown in the figure, these loci are indeed SNPs unique to female Chinese softshell turtles, and therefore can be used as molecular markers to identify the genetic sex of Chinese softshell turtles. To this end, primer 14749-FT was further designed, along with 14749-F and 14749-R, to distinguish between sexes.

[0048] Example 3:

[0049] It has the implementation content of the above embodiments, wherein the specific implementation methods of the above embodiments can be referred to the above description, and the embodiments here will not be described in detail again; however, the difference between the embodiments in this application and the above embodiments is that:

[0050] In this embodiment, the identification and determination of the genetic sex of the Chinese soft-shelled turtle is as follows:

[0051] The technical solution of this patent will be further described in detail below with reference to specific embodiments;

[0052] 1) Primer Design: Based on the disclosed difference in the genome sequence (SNP) between female and male Chinese soft-shelled turtles according to this invention, three primers, 14749-F, 14749-R, and 14749-FT, were designed. Primers 14749-F and 14749-R are positioned on either side of these SNPs, and the sequence bases of 14749-FT are identical to those on the W chromosome. Please refer to [link to relevant documentation]. Figure 1 .

[0053] 2) Extraction of genomic DNA: The skirts of 50 Chinese soft-shelled turtles (16 males and 16 females from the Dongting strain, 3 males and 3 females from the Huangsha strain, 3 males and 3 females from the Taiwan strain, and 3 males and 3 females from the Japanese strain) were randomly cut and placed in 95% alcohol solution and stored at -20℃; Genomic DNA was extracted using a DNA extraction kit and diluted to about 50 ng / μL with EB elution buffer for later use.

[0054] 3) PCR amplification: Primers 14749-F, 14749-R and 14749-FT were used, and the genomic DNA from step 2) was used as the template for PCR amplification.

[0055] PCR reaction system: The total reaction volume is 25 μl. The specific reaction system is as follows: MIX 12.5 μL (2×FineTaqPCR SuperMix(+dye)), DNA template (50 ng / μL) 1 μL, 14749-F (10 μM) 0.3 μL, 14749-R (10 μM) 0.5 μL, 14749-FT (10 μM) 0.7 μL, and finally made up with dd H2O.

[0056] PCR reaction procedure: Falling PCR was used. Pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s; annealing at 69℃, 67℃, and 65℃ for 5 cycles each; annealing at 63℃ for 15 cycles and 30 s each; extension at 72℃ for 30 s; for a total of 30 cycles; extension at 72℃ for 7 min; and finally storage at 4℃.

[0057] 4) Agarose gel electrophoresis detection and result analysis: 5 μL of PCR product was spotted onto a 1.5% ordinary agarose gel (containing gelstain). DNA marker was used as a reference. Electrophoresis was performed at 0.5 V / cm for 1 hour. After electrophoresis, the amplification results were observed and photographed using a gel imaging system. This three-primer combination amplified two specific bands in the genomic DNA of all female individuals, one approximately 401 bp and the other approximately 265 bp, while only one specific band of approximately 401 bp was amplified in the genomic DNA of all male individuals. This is completely consistent with actual conditions. Figure 3 As shown.

[0058] In this embodiment, it is demonstrated that the three primers of the present invention can accurately identify the genetic sex of the Dongting strain of Chinese soft-shelled turtles with an accuracy of 100%. In the verification experiment, we also performed PCR amplification on the genomic DNA of 18 other Chinese soft-shelled turtles from three different regions (3 males and 3 females), and read the gel electrophoresis results. We found that in these 18 turtles, females could amplify two bands of 265bp and 401bp, while males could only amplify one band of 401bp. This indicates that the method can accurately identify the genetic sex of individuals of different strains of Chinese soft-shelled turtles (see...). Figure 4 ).

[0059] In this invention, three specific marker primers were designed. After population validation, it was found that the specific marker primer set can quickly, easily and accurately identify the genetic sex of different strains of Chinese soft-shelled turtle. This lays the foundation for the successful development of sex control breeding of Chinese soft-shelled turtle, the development of monosex farming, and the research on the genome selection breeding and sex determination mechanism of Chinese soft-shelled turtle.

[0060] In this invention, the six SNPs involved are all heterozygous in female individuals and homozygous in male individuals, which can ensure the specific binding of primers and ensure the accuracy of typing.

[0061] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention. <110> Hunan University of Arts and Sciences Hunan Agricultural University <120> A genetic sex-related SNP marker for Chinese soft-shelled turtle, its primers and applications <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 443 <212> DNA <213> Chinese soft-shelled turtle (Pelodiscus sinensis) <400> 1 aatccctgtg atccagggag gagctgctgc tgtagttcat ctccatgctg caggtagaaa 60 gttggtcccc tgaggaagaa gctggcacag gccgacagtc agcacccacc agggcatgcc 120 catgtgccag aggcttgtcc tgctgggggc cctccaggag caggattgcc cccaggtttg 180 tgccaccagc atcaccccca cagcaggggc tcttgctgcg gtaaatataa gggtcatagt 240 cactgctgag agaactacgg aaggtggagc agctcccata gaccccctgg ttgctgacct 300 ctgtacagtc aaccatggaa tcactggagg agcagtggca ctgcccagaa ctgctgctgc 360 tactgtcact gcctgggcag tcagccaagt aaccgctgca cacagagcgg tgaccatgat 420 agcagctgga ggtgctgcca ctt 443 <210> 2 <211> 22 <212> DNA <213> Chinese soft-shelled turtle (Pelodiscus sinensis) <400> 2 gctgctgctg tagttcatct cc 22 <210> 3 <211> 22 <212> DNA <213> Chinese soft-shelled turtle (Pelodiscus sinensis) <400> 3 gctatcatgg tcaccgctct gt 22 <210> 4 <211> 19 <212> DNA <213> Chinese softshell turtle (Pelodiscus sinensis) <400> 4 agcaggattg cccccaggt 19

Claims

1. A genetic sex-related SNP marker for the Chinese soft-shelled turtle, characterized in that, The SNPs are labeled as follows: from the 5' end of the sequence shown in SEQ ID NO:1, the bases at positions 164, 167, 172, 173, 174, and 177 are A / G, C / T, T / C, T / C, G / A, and C / T. Individuals with the SNPs labeled as AG, CT, TC, TC, GA, and CT are female Chinese soft-shelled turtles, and individuals with the SNPs labeled as AA, CC, TT, TT, GG, and CC are male Chinese soft-shelled turtles.

2. A primer set for detecting genetic sex-related SNPs markers of the Chinese soft-shelled turtle as described in claim 1, characterized in that, The nucleotide sequences of the primer set are shown in SEQ ID NO:2-4.

3. A kit for genetic sex determination of Chinese soft-shelled turtles, characterized in that, The kit includes reagents for identifying the SNPs labeled according to claim 1, wherein the reagents include the primer set according to claim 2.

4. A method for identifying the genetic sex of the Chinese soft-shelled turtle, characterized in that, Genetic sex determination of the Chinese soft-shelled turtle by detecting the SNPs markers described in claim 1 includes the following steps: Step 1: Extract genomic DNA from the Chinese soft-shelled turtle to be tested; Step 2: Using a primer set, the genomic DNA of the Chinese soft-shelled turtle to be tested is amplified by PCR to obtain the PCR amplification product; Step 3: Perform electrophoresis on the PCR amplification products to determine the genetic sex of the Chinese soft-shelled turtle based on the number and size of the PCR amplification bands; The primer nucleotide sequences are shown in SEQ ID NO:2-4; the PCR amplification conditions are: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, annealing at 69℃, 67℃, and 65℃ for 5 cycles each, annealing at 63℃ for 15 cycles and 30 s, extension at 72℃ for 30 s, for a total of 30 cycles; extension at 72℃ for 7 min; if the PCR amplification bands are two bands of 265 bp and 401 bp respectively, then the SNPs are labeled as AG, CT, TC, TC, GA, CT genotypes, and the individual is a genetically female Chinese softshell turtle; if the PCR amplification band is a single band of 401 bp, then the SNPs are labeled as AA, CC, TT, TT, GG, CC genotypes, and the individual is a genetically male Chinese softshell turtle.

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