SNP molecular marker for identifying genetic sex of coreius guichenoti and identification method

Abstract

The present application relates to the technical field of fish molecular genetics, and particularly relates to a SNP molecular marker and a method for identifying the genetic sex of Coreius guichenoti. The SNP molecular marker is located in a fragment of a nucleotide sequence shown in SEQ ID NO. 1 in the genome of Coreius guichenoti, and the nucleotides at positions 601, 634, 647 and 650 of the fragment of the nucleotide sequence shown in SEQ ID NO. 1 are SNP sites. By detecting the genotypes of the above-mentioned sites, the accurate identification of male and female individuals of Coreius guichenoti can be realized: when the genotype combination is AA: AA: GG: CC, it is determined to be male; and when the genotype combination is AT: AG: GC: CT, it is determined to be female. The detection system provided by the present application has high specificity and strong stability, and provides an efficient and reliable molecular tool for the artificial breeding, germplasm resource protection and sex determination mechanism research of Coreius guichenoti.

Description

Technical Field

[0001] This invention relates to the field of fish molecular genetics, specifically to SNP molecular markers and identification methods for genetic sex determination in the round-mouthed copper fish. Background Technology

[0002] Round-mouthed bronze fish ( Coreius guichenoti The round-mouthed copper fish (Coreius lumbricoides) belongs to the order Cypriniformes, family Cyprinidae, and genus Coreius. It is a rare and endangered freshwater fish endemic to the upper reaches of the Yangtze River in my country, possessing both extremely high ecological conservation value and potential economic aquaculture value. This species has demanding habitat requirements, and in recent years, its natural population has declined sharply due to factors such as water conservancy projects, water pollution, and overfishing. It has been listed as a Class II protected species in the "National Key Protected Wild Animals List" (2021 edition). Currently, artificial breeding and release of the round-mouthed copper fish have become the core means of its species conservation. However, the aquaculture process faces key problems such as difficulties in early sex identification, low breeding efficiency, and declining population genetic diversity. There is an urgent need to analyze its sex determination mechanism at the genetic level to provide support for precise conservation and the upgrading of aquaculture technology.

[0003] Current research on molecular markers for the round-mouthed copper fish mainly includes the following aspects. In 2018, Xiong Meihua, Shao Ke, and others developed nine microsatellite markers to study and analyze the genetic structure of round-mouthed copper fish populations in the middle and upper reaches of the Yangtze River, showing high levels of genetic diversity in each population. In 2022, He Yongfeng, Zhu Yongjiu, and others analyzed the genetic diversity of round-mouthed copper fish in the middle and lower reaches of the Jinsha River using mitochondrial DNA sequencing technology (such as the COI gene and Cytb), showing no obvious distribution pattern among different geographical populations. Existing technologies rely on traditional markers such as mitochondrial DNA and SSRs, and the research objectives are limited to population genetics, without specific analyses of sex traits, resulting in a complete lack of sex-related genomic information in round-mouthed copper fish. Since research on sex-related markers in the round-mouthed copper fish is still in its infancy, and no candidate markers have been screened, the inherent limitations of current technology suggest the following problems if such research were to be conducted: First, the technology lacks specificity; the development of existing traditional markers has not incorporated sex phenotypic data and cannot be directly used for sex association analysis. Second, the detection efficiency is low; even if sex screening is attempted based on existing SSR markers, a large number of samples must be validated for each marker, resulting in high operational costs and extremely low success rates. The root of these potential shortcomings lies in the fact that current research has not established a "trait-gene" association analysis approach, failing to meet the technical requirements for sex identification. Given the limitations of existing traditional molecular marker technologies in screening sex-related markers for the round-mouthed copper fish, there is an urgent need to develop sex-related SNP molecular markers to solve key problems such as "difficulty in early sex identification, poor marker stability, and unclear sex determination mechanism," and to provide precise molecular technical support for the artificial breeding, release and conservation of the round-mouthed copper fish. Summary of the Invention

[0004] This invention provides an SNP molecular marker and identification method for genetic sex identification of the round-mouthed copper fish, so as to achieve accurate and rapid identification of genetic sex throughout the entire life cycle of the round-mouthed copper fish.

[0005] On one hand, the present invention provides an SNP molecular marker for genetic sex identification of the round-mouthed copper fish. The SNP molecular marker is located in the nucleotide sequence of the round-mouthed copper fish genome as shown in SEQ ID NO.1. The nucleotides at positions 601, 634, 647 and 650 of the nucleotide sequence shown in SEQ ID NO.1 are SNP sites. The polymorphism of the nucleotide at position 601 is A or T, the polymorphism of the nucleotide at position 634 is A or G, the polymorphism of the nucleotide at position 647 is G or C, and the polymorphism of the nucleotide at position 650 is C or T.

[0006] Furthermore, when the genotypes of nucleotides at positions 601, 634, 647, and 650 are AA:AA:GG:CC, the round-mouthed copper fish is male; when the genotypes of nucleotides at positions 601, 634, 647, and 650 are AT:AG:GC:CT, the fish is female.

[0007] On the other hand, the present invention provides a primer pair for genetic sex identification of the round-mouthed copper fish, wherein the primer pair amplifies the above-mentioned SNP molecular marker.

[0008] Furthermore, the primer pair includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.2 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.3.

[0009] On the other hand, the present invention provides a detection reagent for genetic sex identification of the round-mouthed copper fish, the detection reagent being used to detect the above-mentioned SNP molecular markers.

[0010] Furthermore, the detection reagent includes the primer pairs described above.

[0011] On the other hand, the present invention provides a detection kit for genetic sex identification of round-mouthed copper fish, the detection kit comprising the primer pair or the detection reagent described above.

[0012] Furthermore, the detection kit also includes PCR amplification reagents, DNA purification reagents, and / or sequencing reaction buffers.

[0013] On the other hand, the present invention provides the application of the above-mentioned SNP molecular markers or primer pairs or detection reagents or detection kits in identifying the genetic sex of round-mouthed copper fish, preparing products for identifying the genetic sex of round-mouthed copper fish, molecular marker-assisted breeding related to the sex of round-mouthed copper fish, preparing products for molecular marker-assisted breeding of round-mouthed copper fish, genotyping of round-mouthed copper fish, preparing products for genotyping of round-mouthed copper fish, constructing DNA fingerprinting of round-mouthed copper fish and / or preparing products for constructing DNA fingerprinting of round-mouthed copper fish.

[0014] On the other hand, the present invention provides a method for genetic sex determination of the round-mouthed copper fish, the method comprising: detecting the above-mentioned SNP molecular markers in the genome of the round-mouthed copper fish, and determining the sex of the round-mouthed copper fish to be tested based on the genotype of each SNP locus in the detected SNP molecular markers.

[0015] Furthermore, the method includes the following steps: S1. Extract genomic DNA from the fish to be tested, the round-mouthed copper fish; S2. Using the obtained genomic DNA of the round-mouthed copper fish as a template, perform PCR amplification reaction using the primer pairs, detection reagents, or detection kits described above. S3. After the PCR amplification reaction is completed, analyze the sequence information of the PCR amplification product, determine the genotype of the SNP site of the fish to be tested, and identify the sex of the fish to be tested based on the genotype of the SNP site. When the genotypes of nucleotides at positions 601, 634, 647, and 650 are AA:AA:GG:CC, the round-mouthed copper fish is male; when the genotypes of nucleotides at positions 601, 634, 647, and 650 are AT:AG:GC:CT, the fish is female.

[0016] Further, in step S3, the analysis of the sequence information of the PCR amplification product includes: performing one-way Sanger sequencing on the PCR amplification product and using Chromas software to read the sequencing peak diagram of the SNP molecular markers.

[0017] The technical solution of this invention has the following advantages: 1. A new SNP molecular marker for sex-related sex determination in the round-mouthed copper fish was screened for the first time, providing a new perspective for the study of sex determination mechanism in this species.

[0018] 2. A set of PCR amplification primers with high specificity and stability was developed, which can accurately distinguish the sex genotype of the round-mouthed copper fish, providing a reliable technical means for early sex identification.

[0019] 3. Experiments verified the complete correlation (100%) between the selected SNP sites and sex traits, ensuring the accuracy and practicality of the method and filling the gap in the field of sex molecular marker research in the round-mouthed copper fish.

[0020] 4. It provides an efficient molecular tool for the artificial breeding and germplasm resource protection of the round-mouthed copper fish, and has important application value and promotion prospects.

[0021] 5. This approach is not only applicable to the round-mouthed copper fish, but also provides a technical path and research ideas that can be referenced for the study of sex determination mechanisms in other fish species. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 Sequencing profiles for individuals M1, M2, F1, and F2. Detailed Implementation

[0024] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0025] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0026] Example 1: A SNP molecular marker for genetic sex determination in round-mouthed copper fish This embodiment provides an SNP molecular marker for genetic sex identification of the round-mouthed copper fish. The SNP molecular marker is located in the nucleotide sequence of the round-mouthed copper fish genome as shown in SEQ ID NO.1. The nucleotides at positions 601, 634, 647, and 650 of the nucleotide sequence shown in SEQ ID NO.1 are SNP sites. The polymorphism of the nucleotide at position 601 is A or T, the polymorphism of the nucleotide at position 634 is A or G, the polymorphism of the nucleotide at position 647 is G or C, and the polymorphism of the nucleotide at position 650 is C or T.

[0027] When the genotypes of nucleotides at positions 601, 634, 647, and 650 are AA:AA:GG:CC, the round-mouthed copper fish is male; when the genotypes of nucleotides at positions 601, 634, 647, and 650 are AT:AG:GC:CT, the fish is female.

[0028] Example 2: A primer pair for genetic sex determination of the round-mouthed copper fish This embodiment provides a set of primer pairs for sex identification of the roundmouth copper fish based on SNP molecular markers. The primer pairs amplify the SNP molecular markers described in Example 1. The SNP molecular markers are located at bases 601, 634, 647, and 650 from the 5' end of the nucleotide sequence shown in SEQ ID NO.1 of the roundmouth copper fish genome. The primer pairs include an upstream primer with a nucleotide sequence shown in SEQ ID NO.2 (CATCACGCTAGATAACAG) and a downstream primer with a nucleotide sequence shown in SEQ ID NO.3 (ATGGGAAAGCAGAAATA).

[0029] Example 3: A method for genetic sex determination of round-mouthed bronze fish This embodiment provides a method for genetic sex determination of the round-mouthed copper fish based on SNP molecular markers. The steps of the method are as follows: S1. Fins of the round-mouthed copper fish were cut to obtain samples for DNA extraction; genomic DNA was extracted from the round-mouthed copper fish to be tested; genomic DNA was extracted from the fin tissue of the round-mouthed copper fish according to the instructions of the Animal Tissue Genomic DNA Extraction Kit (DP304 type, purchased from Tiangen Biotech (Beijing) Co., Ltd.), and stored at -80°C for later use. S2. Using the obtained genomic DNA of the round-mouthed copper fish as a template, PCR amplification reaction was performed using the primer pair for sex identification of the round-mouthed copper fish in Example 2. The PCR amplification system is as follows: The PCR amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for 35 cycles; 72℃ extension for 10 min. S3. After the PCR amplification reaction is completed, the amplification products are sent to a sequencing company for Sanger sequencing to obtain the peak data and genotype data of the sequencing results; the Sanger sequencing peak data are analyzed to determine whether it is homozygous or heterozygous and to interpret the sex result. When the genotypes of nucleotides at positions 601, 634, 647, and 650 are AA:AA:GG:CC, the round-mouthed copper fish is male; when the genotypes of nucleotides at positions 601, 634, 647, and 650 are AT:AG:GC:CT, the fish is female.

[0030] Example 4: A test kit for genetic sex identification of round-mouthed copper fish This embodiment provides a detection kit for genetic sex identification of the round-mouthed copper fish, the kit comprising the following: The primer pairs, PCR amplification reagents, DNA purification reagents, and sequencing reaction buffers used for sex determination of the roundmouth copper fish in Example 2; The primer pair includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.2 (CATCACGCTAGATAACAG) and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.3 (ATGGGAAAGCAGAAATA).

[0031] Experimental Example 1. Screening of SNP Molecular Markers This experiment aims to screen for a SNP molecular marker for sex association in the croaker (Brassichthys circinus). The experimental procedure is as follows: 1. Selection of test subjects The *Rhizoctonia solani* species selected for this experiment were all from the same strain and were collected from a breeding and conservation base in the Jinsha River, my country. This base's population exhibits high genetic diversity. A total of 52 parent fish samples of *Rhizoctonia solani* (26 females and 26 males) were collected in this experiment. The sample collection process followed a random selection principle to ensure the representativeness of the samples.

[0032] 2. Gender Information Statistics In this experiment, 26 female and 26 male individuals were selected for sample collection. The sex information of the parent fish was determined by dissecting the gonads and observing HE-stained sections of the gonadal tissue. Phenotypic data of sex traits were collected based on the results of dissection and section observation, resulting in tissue samples from 52 parent fish of the round-mouthed copper fish.

[0033] 3. DNA extraction, library construction, and resequencing DNA was extracted from tissue samples of 52 broodstock *Cyprinus circinata* using an animal tissue genomic DNA extraction kit (DP304, purchased from Tiangen Biotech (Beijing) Co., Ltd.). DNA extraction quality was assessed by 0.8% agarose gel electrophoresis, and DNA was quantified using a UV spectrophotometer. Sequencing libraries were prepared using the standard library construction procedure of Illumina's TruSeq DNA PCR-free prep kit. Library quality was assessed using Agilent Bioanalyzer, and quantification was performed using Promega QuantiFluor (≥2 nM). The qualified libraries were denatured with sodium hydroxide and then subjected to paired-end sequencing on the Illumina NovaSeq platform, yielding sequencing data from the 52 broodstock *Cyprinus circinata* tissue samples.

[0034] 4. Mark Filtering Based on resequencing data, SNP molecular markers related to the genetic sex of the round-mouthed copper fish were screened and obtained. These SNP molecular markers are located in the nucleotide sequence of the round-mouthed copper fish genome, as shown in SEQ ID NO.1. Through screening and verification of SNP molecular markers among a large number of male and female round-mouthed copper fish populations, four SNP loci related to the sex of the round-mouthed copper fish were selected and significantly associated with the sex trait (Table 1).

[0035] Table 1. SNP markers showing significant sex-related morphology in the round-mouthed copperfish.

[0036] SEQ ID NO.1: 5. Genotyping Criteria for Sex Identification of Round-Mouthed Bronze Fish Screening and validation results showed that these four candidate loci for sex identification in *Cyprinus circinus* were closely linked in male individuals, with homozygous genotypes in the order AA:AA:GG:CC; and heterozygous loci in female individuals, with genotypes in the order AT:AG:GC:CT. Genotypic statistics for these four loci in different individuals are shown in Table 2.

[0037] Table 2. Statistical information on genotypes at four loci

[0038] To validate the association strength between genotype and sex traits, Phi was calculated using the following formula. )coefficient:

[0039] Among them, a 11 The number of individuals with the genotype AA:AA:GG:CC and who are male; a 12 The number of individuals with the genotype AA:AA:GG:CC and who are female; a 21 The number of individuals with the genotype AT:AG:GC:CT and who are male; a 22 R1 represents the number of individuals with the genotype AT:AG:GC:CT and the sex being female; R2 represents the total number of individuals with the genotype AA:AA:GG:CC; R3 represents the total number of individuals with the genotype AT:AG:GC:CT; C4 represents the total number of individuals with the sex being male; and C5 represents the total number of individuals with the sex being female.

[0040] According to the calculation results, the Phi coefficient for both genotypes is equal to 1, indicating that the genotype is completely associated with sex.

[0041] If the genotypes at these four loci in the sequence are AA:AA:GG:CC (homozygous), then the individual is identified as male; if they are AT:AG:GC:CT, then the individual is identified as female.

[0042] Experimental Example 2: Validation of SNP molecular markers used for sex determination in round-mouthed copper fish This experiment aims to validate SNP molecular markers for sex association in the croaker (Brassichthys circinus). The experimental procedure is as follows: 1. Experimental Materials Thirty-two mature broodstock of the round-mouthed copper fish were randomly selected from the Xiangjiaba Experimental Station of the Yangtze River Biodiversity Research Center of the Three Gorges Group. The sex of the individuals was determined through breeding records, with 16 males and 16 females. Fins of these 32 round-mouthed copper fish were clipped and samples were taken to obtain samples for DNA extraction.

[0043] 2. DNA extraction and amplification Genomic DNA was extracted from the fin tissue of the *Rhizoctonia solani* according to the instructions of the Animal Tissue Genomic DNA Extraction Kit (DP304, purchased from Tiangen Biotech (Beijing) Co., Ltd.), and stored at -80°C for later use. Using the primer pair for sex identification of *Rhizoctonia solani* from Example 2, PCR amplification was performed on 32 individuals of clearly defined physiological sex.

[0044] The PCR amplification system is as follows: The PCR amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for 35 cycles; and 72℃ extension for 10 min.

[0045] The amplified products were sent to a sequencing company for Sanger sequencing to obtain peak data and genotype data from the sequencing results.

[0046] 4. Interpretation of gender identification results The Sanger sequencing peak data were analyzed to determine whether the individuals were homozygous or heterozygous, and the sex was also determined. Genotypes were determined based on sequencing peak data from 32 randomly selected round-mouthed copperfish, using the following criteria: Figure 1 As shown ( Figure 1 (The example only shows the peak plot data of four-tailed individuals M1, M2, F1, and F2). Figure 1 In this study, M1 and M2 were two male individuals, and all four gene loci identified were homozygous (showing a distinct single peak at the locus), in the order A:A:G:C; F1 and F2 were two female individuals, and all four gene loci were homozygous (showing a distinct double peak at the locus), in the order AT:AG:GC:CT. The genotypic statistics of the 32 individuals are shown in Table 3. Table 3: Statistical data on the sequencing genotypes of 32 individuals

[0047] 5. Results Analysis The sex of all the individuals identified based on the sequencing results was consistent with the sex in the breeding records, indicating that the SNP sites screened in this application are completely related to the sex of the round-mouthed copper fish. The primers involved can be used for SNP site genotyping and can be effectively applied to the molecular-level sex identification of the round-mouthed copper fish.

[0048] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A SNP molecular marker for genetic sex determination in the round-mouthed copper fish, characterized in that, The SNP molecular marker is located in the nucleotide sequence of the genotype of the roundmouth copper fish as shown in SEQ ID NO.

1. The nucleotides at positions 601, 634, 647 and 650 of the nucleotide sequence shown in SEQ ID NO.1 are SNP sites. The polymorphism of the nucleotide at position 601 is A or T, the polymorphism of the nucleotide at position 634 is A or G, the polymorphism of the nucleotide at position 647 is G or C, and the polymorphism of the nucleotide at position 650 is C or T.

2. The SNP molecular marker according to claim 1, characterized in that, When the genotypes of nucleotides at positions 601, 634, 647, and 650 are AA:AA:GG:CC, the round-mouthed copper fish is male; when the genotypes of nucleotides at positions 601, 634, 647, and 650 are AT:AG:GC:CT, the fish is female.

3. A primer pair for genetic sex determination of the round-mouthed copper fish, characterized in that, The primer pair amplifies the SNP molecular marker as described in claim 1 or 2.

4. The primer pair according to claim 3, characterized in that, The primer pair includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 2 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.

3.

5. A detection reagent for genetic sex identification of round-mouthed copper fish, characterized in that, The detection reagent is used to detect the SNP molecular markers as described in claim 1 or 2.

6. A detection kit for genetic sex identification of round-mouthed copper fish, characterized in that, The detection kit includes the primer pair as described in claim 3 or 4 or the detection reagent as described in claim 5.

7. The reagent kit according to claim 6, characterized in that, The detection kit also includes PCR amplification reagents, DNA purification reagents, and / or sequencing reaction buffers.

8. The application of the SNP molecular marker of claim 1 or 2, the primer pair of claim 3 or 4, the detection reagent of claim 5, or the detection kit of claim 6 or 7 in identifying the genetic sex of the round-mouthed copper fish, preparing products for identifying the genetic sex of the round-mouthed copper fish, molecular marker-assisted breeding related to the sex of the round-mouthed copper fish, preparing products for molecular marker-assisted breeding of the round-mouthed copper fish, genotyping of the round-mouthed copper fish, preparing products for genotyping of the round-mouthed copper fish, constructing a DNA fingerprint of the round-mouthed copper fish, and / or preparing products for constructing a DNA fingerprint of the round-mouthed copper fish.

9. A method for genetic sex determination of round-mouthed bronze fish, characterized in that, The method includes: detecting the SNP molecular markers of claim 1 or 2 in the genome of the round-mouthed copper fish, and identifying the sex of the round-mouthed copper fish to be tested based on the genotype of each SNP locus in the detected SNP molecular markers.

10. The method according to claim 9, characterized in that, The method includes the following steps: S1. Extract genomic DNA from the fish to be tested, the round-mouthed copper fish; S2. Using the obtained genomic DNA of the round-mouthed copper fish as a template, perform PCR amplification reaction using the primer pair described in claim 3 or 4, the detection reagent described in claim 5, or the detection kit described in claim 6 or 7. S3. After the PCR amplification reaction is completed, analyze the sequence information of the PCR amplification product, determine the genotype of the SNP site of the fish to be tested, and identify the sex of the fish to be tested based on the genotype of the SNP site. When the genotypes of nucleotides at positions 601, 634, 647, and 650 are AA:AA:GG:CC, the round-mouthed copper fish is male; when the genotypes of nucleotides at positions 601, 634, 647, and 650 are AT:AG:GC:CT, the fish is female.

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