Screening and application of group of SNP (Single Nucleotide Polymorphism) sites related to gonad development traits of crassostrea gigas
By combining genome-wide association analysis and U-Net deep learning, SNP sites related to gonadal development in Pacific oysters were screened out, and specific primer pairs were designed for PCR amplification. This solved the problems of accuracy and high throughput in screening gonadal development traits in Pacific oysters and improved breeding efficiency.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-29
- Publication Date
- 2026-03-13
AI Technical Summary
Existing technologies make it difficult to achieve high-throughput and accurate screening of gonadal development traits in Pacific oysters. Traditional methods are cumbersome and easily affected by subjective factors, making it impossible to achieve effective screening in the early stages of growth or in breeding practices.
By combining genome-wide association analysis with U-Net deep learning-based gonadal ratio quantitative phenotypic analysis, we screened out SNP loci combinations that were significantly associated with gonadal development traits. We then designed specific primer pairs for PCR amplification and sequencing to screen individuals with high levels of gonadal development.
It enables early, non-destructive genotyping, improves the efficiency of breeding reproductive traits in Pacific oysters, and can efficiently screen out parents with high levels of gonadal development for constructing superior families and enhancing reproductive performance.
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Figure CN121653265A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular biology and genetic breeding, and relates to the screening and application of a group of SNP molecular markers related to gonadal development traits in the Pacific oyster. Background Technology
[0002] The Pacific oyster is one of the world's most important marine aquaculture shellfish, boasting advantages such as a short food chain, high yield, high profitability, and wide distribution. In artificial breeding, its reproductive performance significantly impacts the success rate of seedling production and aquaculture efficiency. The gonadal tissue of the Pacific oyster is distributed throughout its body, composed of numerous tubules folded within connective tissue containing large sac-like cells. These tissues constitute the gonadal region and undergo significant changes throughout the reproductive cycle, closely related to the cycle of energy storage utilization and other abiotic factors such as photoperiod and temperature. Gonadal development is one of the core reproductive traits; traditional assessment methods rely on anatomical or histological observation, which are cumbersome, have low throughput, and are easily influenced by subjective factors, making accurate, high-throughput screening impossible in early growth stages or breeding practices.
[0003] Genome-wide association studies (GWAS) are an effective method for uncovering key genetic variations in complex traits. However, to date, there is a lack of effective SNP molecular markers in the Pacific oyster that have been validated by large-scale population GWAS and are directly related to gonadal development traits. Using the precise quantitative phenotypic analysis method for gonadal proportion (GP) in Pacific oysters based on U-Net deep learning (see separate application), which was previously established by the inventors, accurate and objective gonadal development phenotypic data from large-scale populations can be obtained. Combining this high-throughput phenotypic data with whole-genome resequencing data for GWAS analysis allows for the precise localization of key molecular markers and corresponding genes related to gonadal development traits.
[0004] Based on this, the present invention is proposed. Summary of the Invention
[0005] Based on the above reasons, the purpose of this invention is to overcome the shortcomings of the prior art, provide a set of SNP loci that are significantly associated with gonadal development traits in Pacific oysters, and provide a method for verification of genome-wide association analysis, screening out 4 SNP loci that can be used to guide the assisted breeding of gonadal development traits in Pacific oysters.
[0006] To achieve the above objectives, the first technical solution of this application discloses a set of SNP markers that are significantly associated with the gonadal development traits of the Pacific oyster, located in two genomic regions, including three SNP sites on the Region 1 fragment and one SNP site on the Region 2 fragment;
[0007] The Region1 fragment is located at positions 51477413-51487927 on chromosome NC_047560.1 of the Pacific oyster, within the BP10 (embryoprotein 10) gene region. The base sequence is shown in SEQ ID No:1. The three SNP sites are located at positions 51,480,280, with mutation types of G / A; positions 51,480,439, with mutation types of A / G; and positions 51,486,796, with mutation types of T / A.
[0008] Region 2 is located at positions 37925281-37938300 on chromosome NC_047565.1 of the Pacific oyster, within the GALNT5 (polypeptide N-acetylgalactosamine transferase 5) gene region. The base sequence is shown in SEQ ID No:2. The SNP site is located at position 37,925,486, and the mutation type is T / C.
[0009] Furthermore, at the three SNP loci within Region 1, the genotypes with the highest gonadal ratio, from highest to lowest, are as follows: SNPs at positions 51, 480, and 280: AA > AG > GG; SNPs at positions 51, 480, and 439: GG > GA > AA; SNPs at positions 51, 486, and 796: AA > AT > TT. At the SNP loci within Region 2, the dominant genotypes associated with a high gonadal ratio are: TT > TC > CC.
[0010] Furthermore, the dominant genotypes at the three SNP molecular marker sites in Region 1 were: SNP at positions 51, 480, and 280, AA type; SNP at positions 51, 480, and 439, GG type; and SNP at positions 51, 486, and 796, AA type. The dominant genotype at the SNP molecular marker sites in Region 2 was TT type.
[0011] The second technical solution of the present invention discloses a specific primer pair for detecting key SNP molecular markers in gonadal development of the Pacific oyster, including the amplification primer pair of the Region1 fragment and the Region2 fragment described in the first technical solution. The forward primer sequence of Region1 is as shown in SEQ ID No:3 and the reverse primer sequence is as shown in SEQ ID No:4; the forward primer sequence of Region2 is as shown in SEQ ID No:5 and the reverse primer sequence is as shown in SEQ ID No:6.
[0012] The third technical solution of this application discloses the application of the key SNP marker combination for gonadal development of the Pacific oyster described in the first technical solution in the breeding of Pacific oysters for gonadal development traits, which can be used to screen Pacific oyster individuals with high gonadal development levels.
[0013] The fourth technical solution of this application discloses a screening method for identifying oyster individuals with a dominant genotype of high gonadal development, comprising the following steps:
[0014] S1. Extract DNA from individual Pacific oysters;
[0015] S2. Amplify oyster DNA sequences using specific primer pairs for key SNP markers of gonadal development traits in oysters;
[0016] S3. Sequencing the amplified oyster DNA sequence to determine the genotype at the four SNP gene loci;
[0017] S4. Select homozygous oysters with two or more dominant genotypes as individuals with high gonadal development.
[0018] Furthermore, the method for amplifying the DNA sequence is PCR amplification reaction, with the following reaction program: reaction at 95 ºC for 3 min; reaction at 95 ºC for 15 s, reaction at 60 ºC for 20 s, reaction at 72 ºC for 1 min, for a total of 35 cycles, followed by reaction at 72 ºC for 5 min.
[0019] Furthermore, the dominant genotypes at the three loci within the Region 1 fragment are: SNP at positions 51, 480, and 280, type AA; SNP at positions 51, 480, and 439, type GG; and SNP at positions 51, 486, and 796, type AA; and at the loci within the Region 2 fragment, type TT.
[0020] The beneficial effects of this invention are as follows:
[0021] This invention utilizes molecular markers identified through large-scale population GWAS that are significantly associated with precise phenotypes (gona ratios). These markers are located within functionally defined candidate genes (BP10, GALNT5). Specific primers and clearly defined dominant allele information are provided, facilitating manipulation and application in routine molecular laboratories, enabling early and non-destructive genotyping. By evaluating the combined genotypes of key SNPs, parents with high potential for gonadal development can be effectively screened for constructing superior families and improving the efficiency of breeding reproductive traits in the Pacific oyster. Attached Figure Description
[0022] Figure 1 shows the genome-wide association analysis of gonadal development in the Pacific oyster: a is the Manhattan plot; b is the Q-Q plot.
[0023] Figure 2 shows the association and genotype effect of the Region 1 locus: a is the localization map of the association signal in the Region 1 region; b, c, and d are the genotype-phenotype effect maps of the three SNP loci in Region 1, respectively.
[0024] Figure 3 Region 2 locus association and genotype effect diagram: a) Region 2 association signal localization diagram; b) Region 2 SNP locus genotype-phenotype effect diagram.
[0025] Detailed Implementation Plan
[0026] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention.
[0027] The first embodiment of this application discloses a set of key SNP markers for gonadal development traits in the long oyster, including three SNP sites on the Region 1 fragment and one SNP site on the Region 2 fragment.
[0028] The three SNP sites in Region 1 are located at positions 51,480,280 (G / A mutation), positions 51,480,439 (A / G mutation), and positions 51,486,796 (T / A mutation); their base sequences are shown in SEQ ID No:1. The SNP site in Region 2 is located at position 37,925,486 (T / C mutation); its base sequence is shown in SEQ ID No:2.
[0029] In this embodiment, the aforementioned SNP marker sites are key sites determining the gonadal development level of the Pacific oyster, and they are obtained through the following screening method: Detailed Implementation
[0030] 1. Identification and Functional Association Analysis of SNP Markers
[0031] S1. Collection of experimental materials:
[0032] In May 2021, 680 12-month-old Pacific oysters were collected in Rongcheng City, Shandong Province. Shell length, shell height, total weight, and shell weight were measured, with length and weight accuracy of 0.01 mm and 0.01 g, respectively. After measurement, the oysters were dissected, and gonads and adductor muscle tissue were collected. The gonad tissue was preserved in paraformaldehyde solution for 24 hours, and then placed in 70% ethanol. Subsequently, the samples were dehydrated, embedded, sectioned, and stained to obtain gonad sections, which were then observed and photographed using an optical microscope to obtain microscopic images. The adductor muscle tissue was flash-frozen in liquid nitrogen and then stored at -80 °C for later use.
[0033] S2. Gonadal ratio calculation:
[0034] Using a precise image segmentation method based on U-Net deep learning, gonadal tissue sections from 680 12-month-old individuals were analyzed to obtain the precise GP value for each individual. The GP values ranged from 3.77% to 99.99%, with an average of 58.78% ± 25.03, exhibiting rich phenotypic variation.
[0035] S3. Construction of whole-genome resequencing libraries:
[0036] From 680 12-month-old individuals, 283 individuals with significant differences in gonadal development phenotype (GP value) were selected as the research sample. DNA sequencing libraries with insert lengths of 400–500 bp were constructed and sequenced using a high-throughput sequencing platform. DNA sample concentration was measured, and DNA sample integrity was assessed using 1% agarose gel electrophoresis. Short DNA fragments meeting the requirements were obtained using transposase and gently mixed to prepare the ligation reaction system. The constructed DNA libraries were first quantified, requiring a concentration >2.3 ng / µl, a total volume >100 ng, and a volume >10 µl. The libraries were checked by agarose gel electrophoresis to ensure a single peak shape and the absence of heterogeneous peaks. After quality control, different libraries were mixed into a flow cell according to the effective concentration and target downstream data volume. Cluster generation was performed, and sequencing was performed on the Illumina NovaSeq 6000 platform.
[0037] S4. SNP identification and genotyping:
[0038] Residual adapter sequences and low-quality reads were removed, and clean reads were then aligned to the reference genome of the Pacific oyster. The SNP genotype for each oyster was estimated using the HaplotypeCaller pipeline. Bioinformatics tools were used to impute missing genotypes and obtain genome-wide SNP genotype data. Further SNP filtering removed low-quality SNPs with imputation scores less than 0.9, deletion rates greater than 10%, and minor allele frequencies less than 0.05, resulting in 4,786,478 high-quality SNPs.
[0039] S5. Genome-wide association study:
[0040] Phenotypic and genotypic data from 283 individuals were used as input for GWAS using a mixed linear model (MLM) in Emmax software. Sex was included as a covariate to account for inter-individual sex differences. Manhattan plots and quantile-quantile plots (QQ plots) were created using the CMplotR package to visualize the GWAS results (see attached). Figure 1 The genome-wide significance threshold was 4.45 × 10⁻⁶. -6 A total of 11 SNPs that were genomically significant with the GP trait were detected.
[0041] Furthermore, this invention screened four SNPs located within key functional genes and exhibiting extremely high correlation as core markers, as shown in Table 1: Region 1 is located on chromosome NC_047560.1 at positions 51477413-51487927, within the BP10 gene region, and contains three SNP sites: at positions 51,480,280 (G / A mutation type), at positions 51,480,439 (A / G mutation type), and at positions 51,486,796 (T / A mutation type); Region 2 is located on chromosome NC_047565.1 at positions 37925281-37938300, within the GALNT5 gene region, and contains one SNP site at position 37,925,486 (T / C mutation type).
[0042] Table 1 SNP sites and mutation types
[0043]
[0044] The results of the association analysis between genetic variation and GP level at the four selected loci are attached. Figure 2 (SNP sites in Region 1) and appendices Figure 3 (SNP sites in Region 2) As shown: At the three SNP sites in Region 1, the mutant individuals with the highest to lowest gonadal ratio are as follows: SNPs at positions 51, 480, and 280, AA>AG>GG; SNPs at positions 51, 480, and 439, GG>GA>AA; SNPs at positions 51, 486, and 796, AA>AT>TT; At the SNP sites in Region 2, the dominant genotypes associated with a high gonadal ratio are: TT>TC>CC.
[0045] In summary, through genotypic analysis at the four SNP loci, the dominant genotypes at the three SNP molecular marker loci in Region 1 were determined to be: SNP at positions 51, 480, and 280, genotype AA; SNP at positions 51, 480, and 439, genotype GG; and SNP at positions 51, 486, and 796, genotype AA. The dominant genotype at the SNP molecular marker loci in Region 2 was determined to be genotype TT.
[0046] The second embodiment of this application discloses a specific primer pair for detecting key SNP molecular markers in gonadal development of the oyster *Crassostrea gigas*, the primer pair comprising:
[0047] 1. Primer pairs for amplifying SNP sites within Region 1 (BP10 gene region, SEQ ID No: 1): including the forward primer as shown in SEQ ID No: 3 of the sequence listing, and the reverse primer as shown in SEQ ID No: 4 of the sequence listing.
[0048] 2. Primer pairs for amplifying SNP sites within Region2 (GALNT5 gene region, SEQ ID No:2): including the forward primer as shown in SEQ ID No:5 and the reverse primer as shown in SEQ ID No:6.
[0049] In this embodiment, the primer pair is used to specifically amplify the genomic DNA extracted from the oyster to be tested, obtaining the corresponding PCR product. The PCR product can be used to analyze the genotype of the oyster DNA at the above-mentioned SNP sites (specifically, 3 sites in Region 1 and 1 site in Region 2) by sequencing or other genotyping techniques, providing a basis for subsequent assessment of the individual's gonadal development potential.
[0050] In a further embodiment, the present application uses agarose gel electrophoresis to detect the PCR product. The results show that the amplified product is a single, bright, specific band, which is consistent with the expected fragment size, indicating that the above primer pair has good specificity and amplification efficiency.
[0051] The third embodiment of this application discloses the application of the key SNP marker combination for gonadal development of the Pacific oyster described in the first embodiment in the breeding of Pacific oyster reproductive traits, specifically it can be used to screen Pacific oyster parents with a high gonadal ratio.
[0052] In breeding practice, by identifying the genotypes of individual oysters on the SNP molecular markers described in the first embodiment, and based on the known association between each genotype and the gonadal ratio, the target genotype required for breeding can be determined. Specifically:
[0053] For loci located within Region 1 (BP10 gene), individuals carrying the dominant homozygous allele (such as the AA type at positions 51,480,280 and 51,486,796, and the GG type at position 51,480,439) are given priority for selection.
[0054] For loci located within Region2 (GALNT5 gene) (locations 37, 925, 486), individuals carrying the dominant homozygous allele (TT type) are preferentially selected.
[0055] By comprehensively evaluating the dominant genotypes of multiple markers, parental individuals with excellent gonadal development potential can be screened efficiently and non-destructively for constructing families with high reproductive performance or breeding new strains, thereby accelerating the genetic improvement process of gonadal development traits in the Pacific oyster.
[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A group of key SNP molecular markers for gonadal development in the long oyster, characterized in that, This includes three SNP sites on the Region 1 fragment and one SNP site on the Region 2 fragment; Region 1 is located at positions 51477413-51487927 on chromosome NC_047560.1 of the Pacific oyster, within the BP10 gene region. Its base sequence is shown in SEQ ID No:1, with three SNP sites: positions 51,480,280, mutation type G / A; positions 51,480,439, mutation type A / G; and positions 51,486,796, mutation type T / A. Region 2 is located at positions 37925281-37938300 on chromosome NC_047565.1 of the Pacific oyster, within the GALNT5 gene region. Its base sequence is shown in SEQ ID No:2, the SNP site is at position 37,925,486, and the mutation type is T / C.
2. The key SNP molecular marker for gonadal development in the long oyster according to claim 1, characterized in that, The genotypes with the highest to lowest gonadal proportions at the three SNP loci within Region1 are as follows: at positions 51, 480, and 280, AA > AG > GG; at positions 51, 480, and 439, GG > GA > AA; and at positions 51, 486, and 796, AA > AT > TT. The genotypes with the highest to lowest gonadal proportions at SNP sites within Region2 are: TT > TC > CC.
3. The key SNP molecular marker for gonadal development in the long oyster according to claim 1, characterized in that, The dominant genotypes at the three SNP loci within the Region1 fragment are as follows: 51, 480, 280, AA; 51, 480, 439, GG; 51, 486, 796, AA. The dominant genotype at the SNP loci within Region2 is the TT type.
4. A specific primer pair for detecting the key SNP molecular marker for gonadal development in the oyster of claim 1, characterized in that, include: The primer pair used to amplify the Region1 fragment has the forward primer sequence shown in SEQ ID No:3 and the reverse primer sequence shown in SEQ ID No:
4. The primer pair used to amplify the Region2 fragment has the forward primer sequence shown in SEQ ID No:5 and the reverse primer sequence shown in SEQ ID No:
6.
5. The application of the key SNP molecular marker combination for gonadal development in the Pacific oyster according to claim 1 in the breeding of Pacific oysters for gonadal development traits, characterized in that, It can be used to screen for individuals of the Pacific oyster with high levels of gonadal development.
6. A screening method for identifying oyster individuals with a dominant genotype of high gonadal development, characterized in that, Includes the following steps: S1. Extract DNA from individual Pacific oysters; S2. Amplify the DNA sequence of *Crassostrea gigas* using the specific primer pair described in claim 4; S3. Sequencing the amplified DNA sequence to determine the genotype at the four SNP sites described in claim 1; S4. Screening for homozygous genotypes containing the dominant genotypes as described in claim 3 to identify oysters with high gonadal development levels.
7. The screening method according to claim 6, characterized in that, The method for amplifying the DNA sequence is a PCR amplification reaction using sequence-specific primers.
8. The screening method according to claim 6 or 7, characterized in that, The dominant genotypes are: AA type at position 51,480,280, GG type at position 51,480,439, and AA type at position 51,486,796 in Region 1; and TT type at position 37,925,486 in Region 2.