Method for accurately evaluating crassostrea gigas germplasm purity

Abstract

The invention discloses a method for accurately evaluating the germplasm purity of crassostrea gigas, and relates to the technical field of aquatic genetic breeding biology, the core of the method is to construct an index based on an NCBI (National Center of Biotechnology Information) crassostrea gigas genome sequence by virtue of a whole genome re-sequencing technology, collect 150 crassostrea gigas and 150 Fujian oysters in a natural population, extract mantle DNA (Deoxyribose Nucleic Acid) for sequencing, screen out 635 specific SNP (Single Nucleotide Polymorphism) sites, and obtain the purity of the crassostrea gigas. The method comprises the following steps: constructing a pure variety feature sequence, sequencing a to-be-detected sample, comparing two detection sequences of 635 sites with the feature sequence, and calculating a consistency average value according to the same base number / sequence length * 100%, namely the germplasm purity value. The method fills the blank that quantitative evaluation cannot be carried out in the prior art, is high in precision and reliability, and can provide key technical support for crassostrea gigas breeding improvement, germplasm protection, industrial standardization and the like.

Description

Technical Field

[0001] This invention relates to the field of aquatic genetic breeding biotechnology, and in particular to a method for accurately assessing the germplasm purity of the Pacific oyster. Background Technology

[0002] The Pacific oyster, a globally farmed shellfish, is a major farmed oyster species in the coastal areas of northern my country. It is naturally distributed in the Northwest Pacific Ocean, including coastal areas of northern China, South Korea, North Korea, and Japan. Pacific oysters are highly nutritious and economically valuable, and are a high-quality seafood that can be eaten raw. As one of the largest farmed oyster species in my country, Pacific oyster farming currently relies almost entirely on artificially bred seedlings. To enhance the high-temperature adaptability of Pacific oyster seedlings, hybridization and backcrossing with the Fujian oyster from southern my country are commonly used in artificial breeding to improve their traits. With the continuous development of Pacific oyster genetic breeding and the increasing demand for diversified quality cultivation, precise control of the genetic background during the breeding process has become increasingly important. To ensure precise control of the genetic breeding process and guarantee the phenotypic stability and aquaculture suitability of the bred strains, it is urgent to establish a method for assessing the germplasm purity of Pacific oysters to promote the high-quality development of Pacific oyster genetic breeding, seedling cultivation, and related industries. On the other hand, given that the artificial cultivation areas of Pacific oysters are adjacent to their natural distribution areas, there is an urgent need for Pacific oyster germplasm fine evaluation technology to monitor the impact of exogenous gene flow on local natural populations, thereby providing technical support for the assessment and protection of the genetic diversity of Pacific oyster natural populations.

[0003] Currently, there is no precise technical method for assessing the germplasm purity of the Pacific oyster. Only techniques for oyster species identification based on oyster genetic or gene information exist, primarily including: Based on differences in the mitochondrial COX1 gene sequence, different species of oysters, such as the Pacific oyster, Fujian oyster, Okinawan oyster, and Hong Kong oyster, can be distinguished. Essentially, this is the identification of different oyster species and does not involve the assessment of the germplasm purity or lineage purity of a single oyster species. Some technologies, using a small number of molecular markers, can identify the F1 generation of hybrids between the Pacific oyster and other oyster species (such as the Fujian oyster), but this does not involve a detailed quantitative assessment of the Pacific oyster's germplasm purity.

[0004] Based on the above, this invention utilizes high-coverage genome resequencing to screen a large number of alleles at highly reliable specific single nucleotide polymorphism sites across the entire genome of the Pacific oyster, counts the number of alleles at specific single nucleotide polymorphism sites in individual Pacific oysters, and calculates the proportion of alleles at purebred loci in the Pacific oyster genome to achieve efficient and accurate quantitative assessment of the germplasm purity of Pacific oysters. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for accurately assessing the germplasm purity of the Pacific oyster. The core idea of ​​this invention is to utilize high-coverage genome resequencing technology to screen a large number of highly reliable alleles of specific single nucleotide polymorphism sites across the entire Pacific oyster genome. By statistically analyzing the number of purebred Pacific oyster-specific SNP alleles in the tested individuals, the pedigree ratio of Pacific oyster-specific alleles is calculated, thereby achieving a precise quantitative assessment of the Pacific oyster germplasm purity.

[0006] Single nucleotide polymorphisms (SNPs) refer to DNA sequence polymorphisms caused by variations in a single nucleotide at the genomic level. They are characterized by their large number, wide distribution, high stability, and ease of detection, making them the most commonly used molecular markers in genetic breeding and germplasm identification. This invention compares the whole genome sequences of the Pacific oyster (Crassostrea gigas) with those of its closely related species (Crassostrea kwangsiensis) to screen for specific SNP loci that appear frequently only in Pacific oysters and infrequently or not at all in Crassula kwangsiensis. These loci can serve as molecular tags to distinguish between Pacific oyster lineage and exogenous lineage. By detecting the allelic types of these specific SNP loci in the tested individuals, the proportion of Pacific oyster lineage can be quantified, enabling precise assessment of germplasm purity.

[0007] To achieve the objectives of this invention, the invention is implemented through the following technical solutions: A method for accurately assessing the germplasm purity of the Pacific oyster includes the following steps: Step 1: Based on the Pacific oyster genome sequence GCA_025765685.3 in the NCBI database, an index was constructed using the bwa program, the samtools program, and the gatk program of the bioinformatics software, respectively. Step 2: Collect natural populations of Pacific oysters and natural populations of Fujian oysters, then extract genomic DNA from the mantle tissue of individual oysters, and finally perform whole-genome resequencing; Step 3: The sequencing data is filtered using the FastP program; Step 4: Use the bwa program to align the resequencing sequences of Pacific oyster and Fujian oyster to the Pacific oyster genome C.gigas.fa, and then use the samtools program to build an index for the bam file; Step 5: Based on the bam file, use the gatk program to generate a genomic variant vcf file; Step 6: Statistical analysis of allele frequencies at single nucleotide variant sites was performed using the vcftools program, followed by identification of specific alleles in the Pacific oyster. Step 7: Obtain 635 single nucleotide polymorphism-specific alleles of the Pacific oyster; Step 8: Extract genomic DNA from the mantle tissue of the oyster individual to be tested and perform whole-genome resequencing; Step 9: Compare the sequencing sequence of the oyster to be tested with the purebred characteristic sequence of the Pacific oyster. Then, calculate the consistency value between the two test sequences and the purebred characteristic sequence of the Pacific oyster according to the preset formula. Finally, take the average of the two consistency values ​​as the germplasm purity value of the Pacific oyster sample to be tested.

[0008] A further improvement is that the sequencing data of each oyster individual is no less than 12G.

[0009] A further improvement is that the screening criterion for the specific allele is that the frequency of the allele in the Pacific oyster population is greater than 96%.

[0010] A further improvement is that the screening criterion for the specific allele is that the frequency of the allele in the Fujian oyster population is less than 5%.

[0011] A further improvement is made by identifying single nucleotide polymorphism sites in the genome according to steps three to five as described above, and then dividing the two alleles of the 635 sites in step 5 in each sample into two detection sequences.

[0012] A further improvement is that the sample size of the natural population of Pacific oyster and the natural population of Fujian oyster is 150 each.

[0013] A further improvement is made in the following formula: Sequence consistency = Number of identical bases ÷ Sequence length × 100%.

[0014] The beneficial effects of this invention are as follows: This invention addresses the limitation of existing technologies, which can only differentiate oyster species or qualitatively identify first-generation hybrids, but cannot quantitatively assess the germplasm purity of the Pacific oyster. It establishes a standardized technical system covering the entire process from genome indexing, sample collection and sequencing, data filtering and comparison, to variant site screening and purity calculation. Precise germplasm purity data clearly defines the genetic consistency of breeding populations, preventing the segregation or degradation of superior traits due to germplasm mixing. This provides accurate pedigree monitoring for Pacific oyster hybridization, backcrossing, and other breeding improvement efforts. The refined germplasm assessment technology provided by this invention can accurately monitor the impact of exogenous gene flow on local natural populations, clearly defining the genetic characteristic boundaries of native germplasm. It provides key technical means for assessing the genetic diversity of natural Pacific oyster populations, protecting superior germplasm resources, and maintaining habitat ecological security, ensuring the integrity and sustainable use of germplasm resources. The unified Pacific oyster germplasm purity assessment standard effectively distinguishes seedlings from purebreds, hybrids, and backcross offspring with different genetic backgrounds. Attached Figure Description

[0015] Figure 1This is a schematic diagram of a method for accurately assessing the germplasm purity of Pacific oysters according to the present invention. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] Accurate assessment of the germplasm purity of Pacific oysters has crucial and irreplaceable benefits for breeding improvement, aquaculture production, germplasm resource protection, industrial standardization, and scientific research applications. Accurate assessment of germplasm purity can clarify the genetic consistency of breeding populations, preventing the segregation or degeneration of superior traits due to germplasm mixing. Pacific oyster seedlings with high germplasm purity exhibit high consistency in key production indicators such as growth cycle, individual size, survival rate, and disease resistance, facilitating unified management during aquaculture. Accurate assessment of germplasm purity can precisely distinguish populations with different genetic backgrounds, clarify the genetic characteristics boundaries of native germplasm, and prevent the dilution or assimilation of superior germplasm resources. Establishing an accurate germplasm purity assessment system allows for the formulation of unified seedling quality standards, regulating the seedling market circulation order, and preventing inferior mixed seedlings from entering the market. Accurate purity assessment can eliminate experimental interference caused by germplasm mixing, ensuring the accuracy, repeatability, and scientific validity of experimental results.

[0018] It should be noted that the technical means not described in detail in the following embodiments are all conventional means in the field, are not the key points of the invention, and will not be elaborated upon.

[0019] Example 1 See Figure 1 This embodiment provides a method for accurately assessing the germplasm purity of the Pacific oyster. Includes the following steps: Step 1: Genome Sequence Index Construction For the reference genome sequence of the Pacific oyster published in the NCBI database, version number GCA_025765685.3, three bioinformatics software programs—bwa, samtools, and gatk—were used to build an index to provide a foundation for subsequent sequence alignment and variant detection. The specific commands are as follows: Use the bwa program to create an index: bwa index C.gigas.fa Use the samtools program to create an index: samtools faidx C.gigas.fa Use the gatk program to create a sequence dictionary: gatk CreateSequenceDictionary -R C.gigas.fa -O C.gigas.dict.

[0020] Step 2: Sample Collection and Whole Genome Resequencing Sample collection: 150 natural populations of Pacific oysters were collected from northern sea areas such as Qingdao, Qinhuangdao, and Dalian, and 150 natural populations of Fujian oysters were collected from southern sea areas such as Ningbo, Xiamen, and Chaozhou. Healthy, undamaged adult individuals were selected to ensure that the samples were representative and covered the genetic diversity of different geographical populations.

[0021] DNA extraction: Genomic DNA was extracted from the mantle tissue of each oyster individual. Whole genome resequencing: High-quality DNA samples are extracted and sent to a sequencing platform for high-coverage whole genome resequencing.

[0022] Step 3: Sequencing data filtering The sequencing data was filtered using the fastp program. The command was: fastp -w 10 -l 150 -q 20 -u40 -n 0 -e 20 -i Sample.raw.R1.fq.gz -I Sample.raw.R2.fq.gz -oSample.R1.fq.gz -O Sample.R2.fq.gz.

[0023] Step 4: Sequence Alignment and BAM File Processing Sequence alignment: The filtered Pacific oyster and Fujian oyster resequencing sequences were mapped to the Pacific oyster reference genome C.gigas.fa using the bwamem program. The specific command was: `bwamem -t 8 -Y -M C.gigas.faSample.R1.fq.gz Sample.R2.fq.gz | samtools sort -@ 7 -o Sample.bam`. Here, `-t8` sets 8 threads, `-Y` enables hard slicing, and `-M` marks reads aligned to multiple positions as secondary alignments to ensure the uniqueness of the alignment results.

[0024] BAM file indexing: The samtools program is used to build an index for the aligned BAM file. The command is: `samtools index Sample.bam`. BAM file indexing can accelerate access to specific regions of the sequence during subsequent variant detection, improving analysis efficiency.

[0025] Step 5: Detection of genomic variant sites Based on the BAM file, use the HaplotypeCaller module of the gatk program to generate a genomic variant VCF (VariantCallFormat) file. The specific command is: `gatk HaplotypeCaller -R C.gigas.fa -ERCGVCF -I Sample.bam -O Sample.vcf.gz`. The `-ERCGVCF` parameter indicates that a genome-wide VCF file is generated, which retains information from all loci, including those where no variants were detected, providing complete data for subsequent allele frequency statistics.

[0026] Step Six: Screening for Specific SNP Sites and Identification of Alleles Allele frequency statistics: The vcftools program was used to count the allele frequencies of SNP loci. The command was: vcftools --vcf Oyster.vcf.gz --keep Sample.txt --freq --out SampleFreq. Here, --keepSample.txt specifies the list of samples to be counted for both the Pacific oyster and the Fujian oyster populations, and --freq indicates the output of allele frequencies. Specific SNP locus screening: Strict screening criteria were set to screen for alleles of purebred Pacific oyster-specific SNP loci: the allele frequency in the Pacific oyster population was >96%, ensuring it was a dominant allele in the Pacific oyster; the allele frequency in the Fujian oyster population was <5%, ensuring it did not originate from exogenous gene contamination from the Fujian oyster.

[0027] Step 7: Construction of purebred characteristic sequences of Pacific oyster Sixty-five single nucleotide polymorphism-specific alleles were obtained from the Pacific oyster, and their coordinates in the Pacific oyster genome (GCA_025765685.3) are as follows: CM056677.2:47545352,CM056677.2:47547400,CM056677.2:47673441,CM056677.2:47673908,CM056677.2:47676220,CM056677.2:47680711,CM056677.2:47708491,CM056677.2:47709266,CM056677.2:47709404,CM056677.2: 47714201,CM056677.2:47723691,CM056677.2:47727120,CM056677.2:4 7748576,CM056677.2:47782003,CM056677.2:47794669,CM056677.2:478 00994,CM056677.2:47802663,CM056677.2:47950155,CM056678.2:5266 9748,CM056679.2:26772443,CM056679.2:34098170,CM056679.2:340983 38,CM056679.2:34099983,CM056679.2:34101972,CM056679.2:3410262 7,CM056679.2:34103760,CM056679.2:34105330,CM056679.2:34106316, CM056679.2:34106850,CM056679.2:34111503,CM056679.2:34111715,C M056679.2:34123963,CM056679.2:34124628,CM056679.2:34124964,CM0 56679.2:34125219,CM056679.2:34126301,CM056679.2:34126688,CM05 6679.2:34127442,CM056679.2:34128058,CM056679.2:34140988,CM0566 79.2:34141138,CM056679.2:34141923,CM056679.2:34144188,CM05667 9.2:34146794,CM056679.2:34147646,CM056679.2:34463100,CM056679.2:35840196,CM056679.2:36041959,CM056679.2:36108423,CM056679.2:36111181,CM056679.2:36114114,CM056679.2:36114428,CM056679.2:36115387,CM056679.2:36116576,CM056679.2:36117982,CM056679.2:36138680,CM056679.2:37609254,CM056679.2:37609843,CM056679.2:38671307 CM056679.2:40418549, CM056679.2:41786690, CM056679.2:41789573, CM056679.2:42740014, CM056679.2:42741902, CM056679.2:42742264, CM056679.2:42742739, CM056679.2:42742859, CM056679.2:47521013, CM056679.2:47656463, CM056679.2:49703836, CM056679.2:49705775, CM056679. 2:49705985,CM056679.2:49706314,CM056679.2:51409396,CM056679.2:51409498,CM056679.2:51432711,CM056679.2:51432953,CM056679.2:51433073,CM056679.2:51433183,CM056679.2:51433184,CM056679.2:51433210,CM056679.2:51433241,CM056679.2:51434373,CM056679.2:51434630 CM056679.2:51434825, CM056679.2:51435412, CM056679.2:51436357, CM056679.2:51436552, CM056679.2:51437111, CM056679.2:51437250, CM056679.2:51440153, CM056679.2:52097642, CM056679.2:52101235, CM056679.2:52103818, CM056679.2:52104149, CM056679.2:52107063, CM056679.2:52348644,CM056679.2:52348925,CM056679.2:52350451,CM056679.2:52350461,CM056679.2:52350462,CM056679.2:52350671,CM056679.2:52350829,CM056679.2:52350842,CM056679.2:52351788,CM056679.2:52351975,CM056679.2:52353346,CM056679.2:53995645,CM056679.2:53995775 CM056679.2:54000380, CM056679.2:54001477, CM056679.2:54001619, CM056679.2:54002295, CM056679.2:54437630, CM056679.2:54454317, CM056679.2:54454398, CM056679.2:55088310, CM056679.2:55088742, CM056679.2:55878152, CM056679.2:55879765, 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[0028] Step 8: Sample processing Genomic DNA was extracted from the mantle of the oyster individuals to be tested, using the same extraction method as in step two to ensure DNA purity and integrity. Subsequently, high-coverage whole-genome resequencing was performed on the extracted DNA. The sequencing platform, library construction method, and data volume requirements were the same as in step two, with a minimum sequencing data volume of 12G per individual to ensure consistency and accuracy of the test results.

[0029] Step 9: Compare the detected sequence with the purebred characteristic sequence of Pacific oyster. Calculate the consistency value between the two detected sequences and the purebred characteristic sequence of Pacific oyster according to the formula. Take the average of the two consistency values ​​as the germplasm purity value of the Pacific oyster sample to be tested. The purebred characteristic sequence of Pacific oyster composed of specific allele nucleotide bases is "".

[0030] The sequencing data volume of each individual oyster is no less than 12G.

[0031] The screening criterion was that the frequency of this allele in the Pacific oyster was greater than 96%.

[0032] The screening criterion was that the frequency of this allele in Fujian oysters was less than 5%.

[0033] Following steps 3-5 above, single nucleotide polymorphism sites in the genome are identified, and the two alleles of the 635 sites in step 5 in each sample are divided into two detection sequences.

[0034] The natural populations of Pacific oysters and Fujian oysters each contained 150 individuals.

[0035] The formula is: Sequence consistency = Number of identical bases ÷ Sequence length × 100%.

[0036] The genome sequence of the Pacific oyster was indexed using the bwa, samtools, and gatk programs. 150 Pacific oysters from northern China and 150 oysters from southern Fujian were collected, and mantle DNA was extracted. High-coverage whole-genome resequencing (≥12G / individual) was performed. The sequencing data was filtered using the FASTP program with clear commands. The resequencing sequences were mapped to the Pacific oyster genome using the bwa program. A BAM file index was created using samtools, and a genomic variant VCF file was generated using gatk. Allele frequencies were statistically analyzed using VCFtools. Pacific oyster-specific alleles with frequencies >96% were identified in Pacific oysters and <5% in Fujian oysters. 635 specific single nucleotide polymorphism sites and their corresponding coordinates were obtained, and a purebred Pacific oyster characteristic sequence was constructed. This invention extracts DNA from oysters for testing, performs ≥12G resequencing, identifies two alleles at 635 loci, forms two detection sequences, calculates the consistency between the two detection sequences and the characteristic sequence, and takes the average value as the germplasm purity value. This breakthrough overcomes the limitations of existing technologies, which can only identify species or first-generation hybrids, and achieves efficient and accurate quantitative assessment of the germplasm purity of the Pacific oyster. It provides technical support for genetic breeding, seedling cultivation, and natural population protection. Existing technologies can only achieve the identification of different oyster species, such as distinguishing between Pacific oysters and Fujian oysters, or the qualitative identification of first-generation hybrids, but cannot quantify the germplasm purity of a single Pacific oyster species. This invention, through whole-genome resequencing and specific locus screening, achieves for the first time an accurate numerical assessment of the germplasm purity of the Pacific oyster, filling a technological gap.

[0037] Test Example 1 Germplasm purity was tested on 10 Pacific oysters taken from an aquaculture farm in Jiaonan, Qingdao.

[0038] Test method: Step 1: Extract DNA from the mantle of these oyster individuals and perform high-coverage whole-genome resequencing. The sequencing data volume of each individual is no less than 12G. For each oyster sample, identify single nucleotide polymorphism sites in the genome according to steps 3-5 above.

[0039] Step 2: Generate two alleles from the 635 loci in Step 7 of Example 1 in each sample into two detection sequences.

[0040] Step 3: Compare the detected sequence with the purebred Oyster oyster sequence in Step 7 of Example 1. Calculate the consistency value between the two detected sequences and the purebred Oyster oyster sequence according to the formula Sequence consistency = Number of identical bases ÷ Sequence length × 100 (%). Take the average of the two consistency values ​​as the Oyster oyster germplasm purity value of the sample to be tested.

[0041] Step 4: After calculation, the purity values ​​of these 10 Pacific oyster germplasm samples are as follows: 99.0446%, 97.1654%, 97.8503%, 99.2126%, 98.8189%, 99.6051%, 98.6614%, 99.0491%, 98.9699%, and 99.5268%.

[0042] Test results: The average purity of these 10 Pacific oyster germplasm samples exceeded 98.5%. The Pacific oyster germplasm purity of this cultured population is extremely high, the genetic background is stable, and it meets the quality requirements for purebred cultured seedlings. The method can accurately capture the genetic characteristics of purebred Pacific oysters, and the test results have small fluctuations, demonstrating high reliability.

[0043] Test Example 2 Germplasm purity of 10 F1 hybrid individuals of Pacific oyster and Fujian oyster was tested. Test method: Step 1: Extract DNA from the mantle of these oyster individuals and perform high-coverage whole-genome resequencing. The sequencing data volume of each individual is no less than 12G. For each oyster sample, identify single nucleotide polymorphism sites in the genome according to steps 3-5 of Example 1.

[0044] Step 2: Generate two alleles from the 635 loci in Step 7 of each sample in Example 1 into two detection sequences.

[0045] Step 3: Compare the detected sequence with the purebred Oyster oyster sequence in Step 7 of Example 1. Calculate the consistency value between the two detected sequences and the purebred Oyster oyster sequence according to the formula Sequence consistency = Number of identical bases ÷ Sequence length × 100 (%). Take the average of the two consistency values ​​as the Oyster oyster germplasm purity value of the sample to be tested.

[0046] Step 4: After calculation, the germplasm purity values ​​of these 10 F1 hybrids of Pacific Ocean oyster and Fujian oyster are as follows: 49.6815%, 49.6800%, 49.9209%, 50.0000%, 49.9213%, 50.0787%, 49.9213%, 50.1577%, 49.9211%, and 49.7627%.

[0047] Test results: The germplasm purity of the 10 F1 hybrid individuals of *Crassostrea gigas* and *Crassostrea kwangsiensis* ranged from 49.6800% to 50.1577%, with an average of close to 50%. The results highly consistent with the genetic theory that the F1 hybrids would contain 50% genetic material from each parent, validating the accuracy of the method. This method can effectively distinguish between hybrid offspring and purebred individuals and can be used for the pedigree purity identification of hybrid seedlings.

[0048] Test Example 3 The germplasm purity of oysters obtained by backcrossing 10 hybrid progeny of Crassula ovata and Crassula fruticosa with Crassula ovata for three generations was tested. Test method: Step 1: Extract DNA from the mantle of these oyster individuals and perform high-coverage whole-genome resequencing. The sequencing data volume of each individual is no less than 12G. For each oyster sample, identify single nucleotide polymorphism sites in the genome according to steps 3-5 above.

[0049] Step 2: Generate two alleles from the 635 loci in Step 7 of each sample into two detection sequences.

[0050] Step 3: Compare the detected sequence with the purebred Oyster moschata sequence from Step 7 above. Calculate the consistency value between the two detected sequences and the purebred Oyster moschata sequence according to the formula Sequence consistency = Number of identical bases ÷ Sequence length × 100 (%). Take the average of the two consistency values ​​as the Oyster moschata germplasm purity value of the sample to be tested.

[0051] Step 4: After calculation, the germplasm purity values ​​of the 10 Pacific oyster hybrids of Fujian oyster and the Pacific oysters after three generations of backcrossing were as follows: 91.1392%, 90.9163%, 93.9968%, 91.1905%, 91.0095%, 90.9455%, 91.5214%, 90.2866%, 93.7898%, and 91.1765%.

[0052] Test results: Germplasm purity values ​​ranged from 90.2866% to 93.9968%, with an average of approximately 91.5%. After three generations of backcrossing, the germplasm purity of the Pacific oyster significantly increased from approximately 50% in the F1 hybrid to over 90%. This method can quantify this purity improvement. The method can accurately assess the effectiveness of pedigree improvement during genetic breeding, providing data support for optimizing breeding programs.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for accurately assessing the germplasm purity of the Pacific oyster, characterized in that: Step 1: Based on the Pacific oyster genome sequence GCA_025765685.3 in the NCBI database, an index was constructed using the bioinformatics software bwa, samtools, and gatk programs, respectively. Step 2: Collect natural populations of Pacific oysters and natural populations of Fujian oysters, then extract genomic DNA from the mantle tissue of individual oysters, and finally perform whole-genome resequencing; Step 3: The sequencing data is filtered using the FastP program; Step 4: Use the bwa program to align the resequencing sequences of Pacific oyster and Fujian oyster to the Pacific oyster genome C.gigas.fa, and then use the samtools program to build an index for the bam file; Step 5: Based on the bam file, use the gatk program to generate a genomic variant vcf file; Step 6: Statistical analysis of allele frequencies at single nucleotide variant sites was performed using the vcftools program, followed by identification of specific alleles in the Pacific oyster. Step 7: Obtain 635 single nucleotide polymorphism-specific alleles of the Pacific oyster; Step 8: Extract genomic DNA from the mantle tissue of the oyster individual to be tested and perform whole-genome resequencing; Step 9: Compare the sequencing sequence of the oyster to be tested with the purebred characteristic sequence of the Pacific oyster. Then, calculate the consistency value between the two test sequences and the purebred characteristic sequence of the Pacific oyster according to the preset formula. Finally, take the average of the two consistency values ​​as the germplasm purity value of the Pacific oyster sample to be tested.

2. The method for accurately evaluating the germplasm purity of the Pacific oyster according to claim 1, characterized in that, The sequencing data of each oyster individual is no less than 12G.

3. The method for accurately evaluating the germplasm purity of the Pacific oyster according to claim 1, characterized in that: The screening criteria for the specific allele is that the frequency of the allele in the Pacific oyster population is greater than 96%.

4. The method for accurately evaluating the germplasm purity of the Pacific oyster according to claim 1, characterized in that: The screening criterion for the specific allele is that the frequency of the allele in the Fujian oyster population is less than 5%.

5. The method for accurately evaluating the germplasm purity of the Pacific oyster according to claim 1, characterized in that: Following steps three to five as described above, single nucleotide polymorphism sites in the genome are identified, and then the two alleles of the 635 sites in step 5 in each sample are combined into two detection sequences.

6. The method for accurately evaluating the germplasm purity of the Pacific oyster according to claim 1, characterized in that: The sample size of the natural population of Pacific oyster and the natural population of Fujian oyster was 150 each.

7. The method for accurately evaluating the germplasm purity of the Pacific oyster according to claim 1, characterized in that: The formula is: Sequence consistency = Number of identical bases ÷ Sequence length × 100%.

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