A method for identifying the gender of melanophryniscus

Abstract

The application relates to a method for identifying the gender of Rana nigromaculata, in particular, the application provides a SNP molecular marker related to the gender of Rana nigromaculata, based on the SNP molecular marker, a gender identification method of Rana nigromaculata and a corresponding reagent system are constructed, and the gender of an individual can be rapidly detected and determined. Compared with the prior art, the application has the advantages of high detection accuracy, simple operation, suitability for early gender identification and the like, can effectively improve the efficiency and accuracy of molecular assisted breeding of Rana nigromaculata, and has a good application prospect.

Description

Technical Field

[0001] This application relates to the field of black-spotted frog technology, and in particular to a method for identifying the sex of black-spotted frogs. Background Technology

[0002] Black-spotted Flyfrog ( Pelophylax nigromaculatus The black-spotted frog (Rana spp.), belonging to the genus Rana of the family Ranidae in the order Anura, is an amphibian also known as the spiny-toothed frog, black-spotted frog, or black frog. It is one of my country's important economically important aquaculture species, possessing advantages such as rapid growth, strong adaptability, and stable market demand, giving it high economic value in aquaculture. However, in actual farming, the sex ratio is difficult to control artificially, and significant differences exist between males and females in growth rate, body size, and economic traits, severely restricting the development of large-scale, intensive black-spotted frog farming. Therefore, establishing efficient and accurate sex identification techniques is of great significance for improving farming efficiency and realizing molecular-assisted breeding.

[0003] Currently, sex identification in black-spotted frogs mainly relies on phenotypic observation or dissection methods. However, these methods are typically only applicable to sexually mature individuals and have drawbacks such as long processing times, complex procedures, and inability to achieve early screening, making it difficult to meet the demands of modern molecular breeding for high-throughput and early identification. Furthermore, while hormone induction and other methods can achieve sex regulation to some extent, their application is limited due to high operational risks, significant environmental impact, and food safety concerns.

[0004] With the development of molecular biology techniques, molecular markers based on single nucleotide polymorphisms (SNPs) have become important tools for trait-related gene localization and molecular-assisted selection due to their high stability, high resolution, and ease of detection. However, stable molecular markers related to sex determination in the black-spotted frog are still relatively lacking, especially SNP sites that can accurately identify sex at an early stage have not yet been systematically developed and applied.

[0005] Therefore, developing a SNP molecular marker that is significantly associated with the sex of the black-spotted frog and establishing a rapid detection method based on this marker is of great theoretical significance and application value for achieving early and accurate sex identification and molecular-assisted breeding of the black-spotted frog. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for identifying the sex of the black-spotted frog.

[0007] The first objective of this invention is to provide the application of reagents for detecting the genotype of SNP sites in the black-spotted frog in the preparation of kits for detecting the sex of the black-spotted frog.

[0008] The second objective of this invention is to provide a reagent for detecting the genotype of SNP sites in the black-spotted frog and its application in detecting the sex of the black-spotted frog.

[0009] The third objective of this invention is to provide a method for detecting the sex of the black-spotted frog.

[0010] The fourth objective of this invention is to provide a kit for detecting the sex of the black-spotted frog.

[0011] The fifth objective of this invention is to provide a product or method for detecting the genotype of SNP loci in the black-spotted frog and its application in the breeding of the black-spotted frog.

[0012] To achieve the above objectives, the present invention is implemented through the following technical solution: This invention claims protection for the application of reagents for detecting the genotype of a SNP locus in the black-spotted frog in the preparation of a kit for sex determination of the frog. The SNP locus is located at bases 238,676,543 of chr1 in the black-spotted frog reference genome ASM5464341v1, and contains three genotypes: CT, CA, and CC. The CC and CA genotypes are female, while the CT genotype is male. The chr1 sequence of the reference genome ASM5464341v1 of the black-spotted frog is available in NCBI, with GenBank accession number CM138083.1.

[0013] The study also included the application of reagents for detecting the genotype of SNP sites in the black-spotted frog in sex determination. The SNP sites are located at bases 238,676,543 of chr1 in the black-spotted frog reference genome ASM5464341v1, and contain three genotypes: CT, CA, and CC. The CC and CA genotypes are female, while the CT genotype is male. The chr1 sequence of the reference genome ASM5464341v1 of the black-spotted frog is available in NCBI, with GenBank accession number CM138083.1.

[0014] Preferably, the reagent for detecting the genotype of the SNP site in the black-spotted frog is a primer with a nucleotide sequence as shown in SEQ ID NO: 1-2, wherein the SNP site is located at 273 bases in the amplification product.

[0015] This invention also claims a method for detecting the sex of the black-spotted frog, specifically by detecting the genotype at the SNP site at bases 238,676,543 of chr1 in the black-spotted frog reference genome ASM5464341v1; wherein the CC and CA genotypes are female, and the CT genotype is male. The chr1 sequence of the reference genome ASM5464341v1 of the black-spotted frog is available in NCBI, with GenBank accession number CM138083.1.

[0016] Preferably, PCR amplification is performed using primers with nucleotide sequences as shown in SEQ ID NO:1-2 to detect the genotype of the SNP site.

[0017] Preferably, the PCR amplification reaction system comprises: 25.0 μl of 2×Rapid Taq Master Mix, 0.0 μl of ddH2O2, 2.0 μl each of primers with nucleotide sequences as shown in SEQ ID NO:1-2, and 1.0 μl of template DNA. Preferably, the PCR amplification reaction program is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, 60℃ annealing for 15 sec, 72℃ extension for 60 sec, for 35 cycles.

[0018] This invention also claims a kit for sex determination in the black-spotted frog, containing reagents for detecting the genotype of a SNP locus in the frog, said SNP locus being located at base 238,676,543 of chr1 in the black-spotted frog reference genome ASM5464341v1, with three genotypes: CT, CA, and CC; wherein the CC and CA genotypes are female, and the CT genotype is male. The chr1 sequence of the reference genome ASM5464341v1 of the black-spotted frog is available in NCBI, with GenBank accession number CM138083.1.

[0019] Preferably, the reagent for detecting the genotype of the SNP site of the black-spotted frog is a primer with a nucleotide sequence as shown in SEQ ID NO: 1-2.

[0020] This invention also claims protection for the application of products or methods for detecting the genotype of SNP loci in the breeding of the black-spotted frog, wherein the SNP locus is located at base 238,676,543 of chr1 in the black-spotted frog reference genome ASM5464341v1, and contains three genotypes: CT, CA, and CC; wherein the CC and CA genotypes are female, and the CT genotype is male. The chr1 sequence of the reference genome ASM5464341v1 of the black-spotted frog is available in NCBI, with GenBank accession number CM138083.1.

[0021] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a SNP molecular marker related to the sex of the black-spotted frog. Based on this SNP molecular marker, a method and corresponding reagent system for sex identification of black-spotted frogs are constructed, enabling rapid detection and determination of individual sex. Compared with existing technologies, this invention has advantages such as high detection accuracy, simple operation, and suitability for early sex identification. It can effectively improve the efficiency and accuracy of molecular-assisted breeding of black-spotted frogs and has good application prospects. Attached Figure Description

[0022] Figure 1 This is the sex QTL interval of the black-spotted frog located by QTLseq (Fst=0.23 is the empirical genomic level threshold).

[0023] Figure 2 To screen the genotype frequency map of SNP loci most significantly associated with sex by statistical analysis of genotype and phenotypic sex data. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0025] Example 1: QTL-seq Identification of Sex-Related Loci I. Construction of the Black-spotted Frog Family and Sex Identification 1. Experimental Methods In March 2024, healthy and high-quality black-spotted frogs were selected from the South China Botanical Garden in Guangdong Province. Pelophylax nigromaculatus One pair of parents were used to construct a full-sib family. The experimental individuals were raised under standard breeding conditions at the South China Botanical Garden in Guangzhou, and a total of 138 offspring individuals were obtained.

[0026] After the offspring reach gonadal maturity, phenotypic sex is determined by artificially observing secondary sexual characteristics such as the crop. Subsequently, toe tissue samples are taken from the offspring, preserved in anhydrous ethanol, and stored at -4°C for later use.

[0027] 2. Experimental Results After sex determination, there were 76 male offspring and 62 female offspring.

[0028] II. Extraction of DNA from the Black-spotted Frog DNA was extracted from 138 progeny individuals using the Novizan Genomic DNA Extraction Kit, following the kit's instructions. The specific method is as follows: a. Take 1–1.5 cm 2 Frog toes were cut into small pieces with sterile scissors, placed on filter paper to absorb residual ethanol, and then placed in lysis buffer (220 μl Buffer ATL and 20 μl Proteinase K) and digested with shaking at 55 °C and 105 r / min for 12 hours. b. After the tissue has been fully lysed, transfer the supernatant to a new centrifuge tube, add 10 μl of RNase A to the digestion solution, let stand for 10 minutes, and then load 250 μl of Buffer DL. c. Continue digestion at 70°C for 1.3 hours, transfer the supernatant, add 250g of anhydrous ethanol and vortex for 15 seconds; d. Pack the elution column into a 2ml collection tube, transfer the liquid into the collection tube, and centrifuge at 12000 × g for 1 minute; e. Discard the filtrate, put the beads back into the collection tube, add 500 μl of Buffer GW1 to the column, and centrifuge at 12000 × g for 1 minute; f. Discard the filtrate, put the beads back into the collection tube, add 650 μl of Buffer GW1 to the column, and centrifuge at 12000 × g for 1 minute; g. Discard the filtrate, put the beads back into the collection tube, and centrifuge at 12000 × g for 2 minutes; h. Place the column into a new 1.5ml centrifuge tube, add 50μl of water preheated to 70℃, let stand for 3 minutes, and centrifuge at 12000× g for 1 minute; i. Add 40 μl of water preheated to 70°C, let stand for 3 minutes, and centrifuge at 12000 × g for 1 minute; g, 1% agarose gel electrophoresis, 120V for 30 min, and photographed using a gel imaging system.

[0029] III. QTL-seq sequencing and trait mapping 1. Experimental Methods Fifty male and fifty female individuals were selected from the offspring population, and equal amounts of DNA (10 μl) were collected from each to construct male-mixed and female-mixed pools, respectively. Whole-genome sequencing was performed on the mixed pool samples.

[0030] After quality control of the sequencing data, the data were aligned to a self-constructed *Rana maculatus* genome (unpublished data) using Bowtie2 software. Subsequently, PoPoolation2 software was used to analyze allele frequency differences between male and female populations, and the FST value across the entire genome was calculated using a sliding window method. Parameters were set as follows: min-qual = 20, min-count = 5, max-coverage = 200, window-size = 5000, step-size = 2500. FST data corresponding to windows with fewer than 40 SNPs were removed. Genomic regions with significantly elevated FST were designated as candidate sex-linked regions based on empirical thresholds; SNP sites with significant allele frequency differences between mixed male and female populations were further screened as candidate sex-linked markers.

[0031] 2. Experimental Results A total of 427,859 Fst value sliding windows were analyzed across the entire genome. The results are shown below. Figure 1 A large number of single nucleotide polymorphisms (SNPs) significantly exceeding the red threshold line (top 1 / 10000 peak, corresponding to an Fst value of 0.23) were clustered in the chr1 region, and the Fst peak value in this region was significantly higher than that in other chromosomal regions. The top 43 highly differentiated peaks with the highest Fst values ​​were all located on chromosome 1 (chr1): chr1:118,572,500–705,610,000, with the highest peak located at chr1:481,377,500. The results indicate that this region is a major QTL region regulating sex differentiation in the black-spotted frog.

[0032] Example 2: SNP marker development and population analysis I. Experimental Methods 1. Primer design Based on the sex-related QTL intervals obtained through screening, candidate gene regions were located in the NCBI database, and specific primers were designed as follows: F: 5'-AAGACCACAGTGCCAACCAC-3' (SEQ ID NO. 1); R: 5'-GACGTCACAGCTTAGCCAAC-3' (SEQ ID NO. 2).

[0033] 2. PCR amplification and sequencing DNA extracted from 138 individuals of the constructed pedigree was subjected to PCR amplification. The reaction system is shown in Table 1. Table 1

[0034] Reaction procedure:

[0035] Steps 2 to 4 are repeated 35 times.

[0036] DNA concentration was determined using a Qubit quantitative PCR instrument and detected using the Qubit dsDNA HS Assay Kit, calibrated according to Standard 1 and Standard 2. Based on the raw DNA concentration (>20 ng / μl) of each sample and the DNA quality shown by 1% agarose gel electrophoresis results, such as high main band quality and no obvious RNA contamination, samples from 98 male and female frogs were obtained that met the requirements for subsequent experiments.

[0037] The specific measurement steps are as follows: Add 198 μl of working solution and 2 μl of DNA sample to each reaction system, and measure its diluted concentration; obtain the original DNA concentration by multiplying the diluted DNA concentration of the sample by the dilution factor (20). If the original DNA concentration of the sample is too high, it is necessary to dilute the sample to control the measured concentration within 50-100 ng / μl, so as not to exceed the measurable range of the fluorescence quantitative PCR instrument.

[0038] PCR products were sent to a sequencing company for Sanger sequencing to obtain ab1 format files. The sequencing data were assembled and analyzed using DNASTARLasergene SeqMan software. Genotypes of each individual were determined based on software analysis and manual interpretation of the sequencing peak diagrams, and the genotype distribution of male and female individuals was recorded.

[0039] II. Result Interpretation The amplification product is shown in SEQ ID NO.3. Statistical analysis of genotype and phenotypic sex data revealed one SNP site located at base 563,871,717 of chr1, corresponding to base 238,676,543 of chr1 (CM138083.1) in the reference genome ASM5464341v1 of the black-spotted frog (Black-spotted Flyfrog) in the NCBI database.

[0040] Three genotypes, CT, CA, and CC, were found in the analyzed population. Males were predominantly heterozygous for CT (46 / 49, 93.9%), with very few CC individuals and none for CA. Females were predominantly homozygous for CC (43 / 49, 87.8%), with very low percentages of CT and CA individuals.

[0041] Statistical analysis showed a highly significant association between the genotype of this SNP and sex (χ2=72.062, df=2, P<0.001). This SNP genotype is highly linked to the sex of the black-spotted frog, with CC biased towards females and CT biased towards males.

Claims

1. Use of a reagent for detecting the genotype of a SNP site of Pelophylax nigromaculatus in the preparation of a kit for detecting the gender of Pelophylax nigromaculatus, characterized in that, The SNP locus is located at bases 238,676,543 of chr1 in the ASM5464341v1 reference genome of the black-spotted frog, and contains three genotypes: CT, CA, and CC. The CC and CA genotypes are female, while the CT genotype is male. The chr1 sequence of the reference genome ASM5464341v1 of the black-spotted frog is available in NCBI, with GenBank accession number CM138083.

1.

2. The application of a reagent for detecting the genotype of SNP loci in the black-spotted frog in the determination of sex in the black-spotted frog, characterized in that, The SNP locus is located at bases 238,676,543 of chr1 in the ASM5464341v1 reference genome of the black-spotted frog, and contains three genotypes: CT, CA, and CC. The CC and CA genotypes are female, while the CT genotype is male. The chr1 sequence of the reference genome ASM5464341v1 of the black-spotted frog is available in NCBI, with GenBank accession number CM138083.

1.

3. The application according to claim 1 or 2, characterized in that, The reagent used to detect the genotype of the SNP site in the black-spotted frog is a primer with a nucleotide sequence as shown in SEQ ID NO:1-2.

4. A method for detecting the sex of the black-spotted frog, characterized in that, Genotypes were detected at the SNP sites at 238,676,543 of chr1 in the ASM5464341v1 genome of the black-spotted frog; the CC and CA genotypes were female, and the CT genotype was male. The chr1 sequence of the reference genome ASM5464341v1 of the black-spotted frog is available in NCBI, with GenBank accession number CM138083.

1.

5. The method according to claim 4, characterized in that, PCR amplification was performed using primers with nucleotide sequences as shown in SEQ ID NO:1-2 to detect the genotype of the SNP site.

6. The method according to claim 5, characterized in that, The PCR amplification reaction system consisted of: 25.0 μl of 2×Rapid TaqMaster Mix, 20.0 μl of ddH2O, 2.0 μl each of primers with nucleotide sequences as shown in SEQ ID NO:1-2, and 1.0 μl of template DNA.

7. The method according to claim 5, characterized in that, The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, 60℃ annealing for 15 sec, 72℃ extension for 60 sec, for 35 cycles.

8. A kit for detecting the sex of the black-spotted frog, characterized in that, The reagent contains a reagent for detecting the genotype of a SNP locus in the black-spotted frog. The SNP locus is located at base 238,676,543 of chr1 in the black-spotted frog reference genome ASM5464341v1, and contains three genotypes: CT, CA, and CC. The CC and CA genotypes are associated with females, while the CT genotype is associated with males. The chr1 sequence of the reference genome ASM5464341v1 of the black-spotted frog is available in NCBI, with GenBank accession number CM138083.

1.

9. The reagent kit according to claim 8, characterized in that, The reagent used to detect the genotype of the SNP site in the black-spotted frog is a primer with a nucleotide sequence as shown in SEQ ID NO:1-2.

10. The application of a kit or method for detecting the genotype of SNP loci in the black-spotted frog in the breeding of the black-spotted frog, characterized in that, The SNP locus is located at bases 238,676,543 of chr1 in the ASM5464341v1 reference genome of the black-spotted frog, and contains three genotypes: CT, CA, and CC. The CC and CA genotypes are female, while the CT genotype is male. The chr1 sequence of the reference genome ASM5464341v1 of the black-spotted frog is available in NCBI, with GenBank accession number CM138083.1.

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