Method for identifying DNA fragments of male parent nibea albiflora in gynogenesis pseudosciaena crocea genome

Abstract

The invention discloses a method for identifying DNA (deoxyribonucleic acid) fragments of male parent nibea albiflora in a gynogenesis pseudosciaena crocea genome. According to the scheme, the pseudosciaena crocea genome is used as a reference coordinate system; male parent positive sites in a gynogenesis pseudosciaena crocea genome are screened by identifying the specific variation sites of the nibea albiflora and analyzing the genotypes of the gynogenesis pseudosciaena crocea at the specific variation sites of the nibea albiflora; performing multi-sequence comparison, calculating the number of positive sites in comparison blocks and the proportion, filtering candidate segments and the like to establish the method for identifying the genetic material from the male parent nibea albiflora in the gynogenetic large yellow croaker genome. At present, related identification is mainly carried out through comparative analysis and microsatellite marking; the methods have the problems of high cost, low positive rate and the like; according to the scheme, genetic materials derived from male parent nibea albiflora in a gynogenetic pseudosciaena crocea genome are comprehensively and accurately screened on the whole genome level, the economic character source of gynogenetic progeny can be defined, and application such as variety identity identification and variety right protection is facilitated by developing specific molecular markers.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and genetic breeding technology, and in particular to a method for identifying paternal yellow croaker DNA fragments in the genome of gynogenetic large yellow croaker. Background Technology

[0002] Large yellow croaker (Larimichthys crocea) is an important fishery resource. With the expansion of large yellow croaker farming scale and production, many problems have emerged in its industrial development, such as germplasm degradation, frequent diseases, and a lack of superior varieties. These problems seriously restrict the sustainable development of the large yellow croaker industry, making it urgent to cultivate new varieties of large yellow croaker with high resistance and quality. Gynogenesis is an important breeding method, and many new aquatic varieties, including "Jian carp," "Changfeng silver carp," "Zhongke Jiayu No. 1," "Beiping No. 1" flounder, "Penglai Red" scallop, and "Xiangyun crucian carp No. 2," have utilized gynogenesis technology. Currently, gynogenic offspring are mainly obtained through the induction of inactivated heterologous sperm. During gynogenesis, genetic material from the paternal sperm enters the genome of the gynogenic offspring. This results in the gynogenic offspring carrying part of the paternal genetic material and often exhibiting some of the paternal phenotypes, showing superior performance in many economic traits, i.e., the "heterospermia effect." Identifying these heterologous fragments can not only clarify the source of superior traits in gynogenetic offspring, but also serve as molecular markers for variety identification and variety rights protection.

[0003] Currently, there are two main methods for identifying paternal DNA in the genomes of gynogenetic offspring:

[0004] 1. Read Depth-Dependent Comparative Analysis (RDC) Prediction. This technique identifies specific DNA fragments in target samples by comparing the read depth of sequencing data from different samples on the reference genome of gynogenetic offspring. The method first aligns high-depth (40× or higher) next-generation sequencing data from the paternal, maternal, and gynogenetic offspring genomes with the assembled, high-quality gynogenetic offspring genome. The read depth of each locus on the reference genome is calculated, and loci meeting the criteria of >20× read depth in both gynogenetic offspring and the paternal genome, and 0 read depth in the maternal genome, are selected as positive loci. Continuous loci form fragments, and fragments less than 1 kb apart are assembled into larger candidate fragments. Finally, specific primers (SCAR markers) are designed for these candidate fragments, and PCR amplification is performed using the paternal, maternal, and gynogenetic offspring genomes as templates to experimentally verify the candidate fragments.

[0005] 2. Microsatellite marker screening. Microsatellites (Simple Sequence Repeats, SSRs) are short tandem repeat sequences of 2-6 bases in the genome, exhibiting high polymorphism and species specificity. Because the number of repeats varies among different species / individuals, the length of the fragments amplified by PCR differs. When a paternal-specific band appears in gynogenetic offspring, it confirms the infiltration of paternal DNA fragments. This method first requires the development of satellite primers. Using these primers, PCR amplification is performed on the paternal, maternal, and gynogenetic offspring. The amplified products are separated by polyacrylamide gel electrophoresis. The microsatellite bands of the paternal, maternal, and gynogenetic offspring are compared. If a paternal-specific band is found in the gynogenetic offspring, that band is recovered and sequenced. Sequence homology is compared to confirm the fragment's origin.

[0006] However, both RDC and microsatellite marker methods have many drawbacks. The main disadvantages of RDC are its high cost and low positive rate. First, RDC relies on high-quality whole-genome data assembled from gynogenetic offspring, as well as high-depth (40× or higher) next-generation sequencing data from the paternal, maternal, and gynogenetic offspring. Especially when identifying exogenous DNA fragments in the genomes of multiple gynogenetic offspring, whole-genome data for each offspring is required, resulting in very high costs. Second, the positive rate of candidate fragments obtained by the RDC method is low; SCAR marker detection revealed that only 25% (12 / 48) of the candidate fragments truly originated from the paternal parent.

[0007] Furthermore, the main drawbacks of microsatellite marker screening are low detection site coverage, high cost, and operational complexity. First, microsatellites are sparsely and unevenly distributed throughout the genome, typically one site every 10-100 kb. Differences in the flanking sequences of microsatellites between parents can hinder the development of specific primers and cause false positives. These problems mean that only a few dozen pairs of microsatellite primers are often used in practice. Therefore, the detected sites represent only a small portion of the entire genome and cannot reflect the integration of exogenous DNA at the whole-genome level. Second, the development of microsatellite markers and the screening of heterologous DNA fragments are time-consuming and labor-intensive, requiring primer design, PCR amplification, polyacrylamide gel electrophoresis, fragment recovery, and sequencing. Moreover, this technology is inefficient. For example, Liao et al. used 60 pairs of microsatellite primers to screen DNA fragments from the paternal mandarin fish in the genome of gynogenetic snakehead, but only one fragment was confirmed. Therefore, the microsatellite marker method cannot comprehensively analyze the integration of exogenous DNA in the genome of gynogenetic offspring. Furthermore, the development of microsatellite markers and the screening of heterologous DNA fragments are time-consuming and labor-intensive.

[0008] Based on the background technology, this method establishes a method for identifying heterologous DNA fragments in the genome of gynogenetic large yellow croaker at the whole genome level. Compared with existing methods, this invention has the advantages of low cost, high positive rate and simple operation, providing technical support for the identification and variety rights protection of gynogenetic large yellow croaker. Summary of the Invention

[0009] In view of this, the purpose of this invention is to provide a method for identifying paternal yellow croaker DNA fragments in the genome of gynogenetic large yellow croaker, which has high coverage, low cost, and is simple and reliable to operate.

[0010] To achieve the above-mentioned technical objectives, the technical solution adopted by this invention is as follows:

[0011] A method for identifying paternal yellow croaker DNA fragments in the genome of gynogenetic large yellow croaker includes: screening for positive sites derived from paternal yellow croaker by identifying the genotype of gynogenetic large yellow croaker at yellow croaker-specific mutation sites; detecting DNA fragments derived from paternal yellow croaker by multiple sequence alignment and calculating the number and proportion of positive sites in the alignment blocks; and confirming the identification by sequencing or SCAR marker detection technology.

[0012] As one possible implementation method, the identification method of this scheme further includes the following steps:

[0013] 1. Data Preparation. Prepare the genome sequences of large yellow croaker and yellow croaker, as well as second-generation resequencing data for common large yellow croaker, gynogenetic large yellow croaker, and yellow croaker. This step requires the large yellow croaker genome sequence to be assembled to the chromosome level in FASTA format, and the second-generation resequencing data to have an average sequencing depth of 10× or higher in FASTA format.

[0014] 2. Quality control of second-generation sequencing data. All second-generation resequencing data underwent quality control, with reads containing Q < 20, N > 10%, and a length < 75 bp removed, as well as polyG / polyX sequences.

[0015] 3. Sequence alignment. All quality-controlled second-generation resequencing data were aligned one by one with the large yellow croaker genome, and only high-quality alignments were retained.

[0016] 4. Repeat sequence marking. An index is built for the alignment file, and repeat sequences are marked.

[0017] 5. Identification of specific variant sites in yellow croaker. Genome typing was performed on both large yellow croaker and yellow croaker populations. Biallelic loci showing variation in yellow croaker but not in large yellow croaker were identified as specific variant sites in yellow croaker.

[0018] 6. Genotyping of gynogenetic large yellow croaker at croaker-specific mutation sites. To reduce computational load, targeted genotyping was performed on gynogenetic large yellow croaker at croaker-specific mutation sites.

[0019] 7. Add croaker-specific variation information to the gynogenetic large yellow croaker genotyping file.

[0020] 8. Screening for paternal positive loci in the genome of gynogenetic large yellow croaker. Loci meeting the following conditions are considered paternal positive loci: 1) The genotype of the gynogenetic large yellow croaker at the croaker-specific variant locus is heterozygous (0 / 1) or homozygous variant (1 / 1); 2) The variant allele of the gynogenetic large yellow croaker at the croaker-specific variant locus is the same as the croaker-specific variant allele; 3) The total depth (DP) is required to be ≥5, and for heterozygous types, the depth of the variant allele is required to be ≥2. For homozygous variant types, the depth of the reference allele is required to be 0.

[0021] 9. Positive sites were merged into continuous candidate DNA fragments. Based on the location information of positive sites in gynogenetic large yellow croaker in the reference genome coordinate system of large yellow croaker, adjacent positive sites were merged into continuous candidate fragments (maximum gap 1Kb), while short fragments (>1kb) were filtered out.

[0022] 10. Obtaining the variant sequence. The variant sequence was obtained by replacing the bases at the positive sites of the candidate fragments with the specific variant bases of the yellow croaker.

[0023] 11. Alignment of variant sequences with the yellow croaker genome. The variant sequences were aligned with the yellow croaker genome using BLASTN software.

[0024] 12. Perform multiple sequence alignment and filter candidate fragments based on the number and proportion of positive sites within the alignment block. Each blastn hit is considered an alignment block. Extract the reference sequence of the yellow croaker (Sequence 1), the variant sequences of candidate fragments within the block (sequence 2, where the positive sites have been replaced with yellow croaker-specific variant bases), and the reference sequence of the large yellow croaker candidate fragment (sequence 3, where the positive sites have not been replaced with yellow croaker-specific variant bases). Perform multiple sequence alignment on these three sequences using MAFFT. If at a variant site, sequences 1 and 2 have the same bases but differ from sequence 3, then that site is a positive site. If at a variant site, sequences 2 and 3 have the same bases but differ from sequence 1, then that site is a non-positive site. Calculate the proportion of positive sites within the block, i.e., number of positive sites / (number of positive sites + number of non-positive sites). Exclude candidate fragments with a positive site proportion <30% or ≤2 positive sites within the block.

[0025] 13. Sequencing Validation and SCAR Marker Detection of the Yellow Croaker Fragment in the Gynogenetic Large Yellow Croaker Genome. Universal primers were designed in conserved regions of the large yellow croaker and yellow croaker sequences. PCR amplification was performed using the genomes of common large yellow croaker, gynogenetic large yellow croaker, and yellow croaker as templates. The amplified products were sequenced, and candidate fragments were validated by multiple sequence alignment. Simultaneously, yellow croaker-specific primers were designed in yellow croaker-specific variant sites. PCR amplification was performed using the genomes of common large yellow croaker, gynogenetic large yellow croaker, and yellow croaker as templates to detect the SCAR marker in gynogenetic large yellow croaker.

[0026] In summary, the process includes: preparing the genome sequences of large yellow croaker and yellow croaker, as well as second-generation resequencing data of common large yellow croaker, gynogenetic large yellow croaker, and yellow croaker; then performing quality control on the second-generation resequencing data, comparing all second-generation resequencing data with the large yellow croaker genome sequence one by one, then labeling repetitive sequences and identifying yellow croaker-specific variant sites, then performing targeted genotyping of gynogenetic large yellow croaker at the yellow croaker-specific variant sites, and adding yellow croaker-specific variant information to the gynogenetic large yellow croaker genotyping file; then screening paternal positive sites in the gynogenetic large yellow croaker genome, then merging positive sites into continuous candidate fragments, then replacing the bases in the positive sites of the candidate fragments with yellow croaker-specific variant bases to obtain variant sequences, then comparing the variant sequences with the yellow croaker genome, filtering candidate fragments through multiple sequence alignment and based on the number and proportion of positive sites in the alignment block, and finally performing sequencing verification and SCAR marker detection on the yellow croaker fragment in the gynogenetic large yellow croaker genome.

[0027] This scheme uses the large yellow croaker genome as a reference coordinate system. It establishes a precise identification method for paternal-derived genetic material in the genome of gynogenetic large yellow croaker by identifying specific variant sites in the croaker and analyzing the genotypes of gynogenetic large yellow croaker at these specific variant sites. Further steps include multiple sequence alignment, calculation of the number and proportion of positive sites within aligned blocks, and filtering candidate fragments. Gynogenesis is an important breeding method, currently mainly obtained through inactivated allogeneic sperm induction. During gynogenesis, genetic material from the paternal sperm enters the genome of the gynogenetic offspring, affecting their economic traits, i.e., the "aliensperm effect." Currently, the identification of paternal genetic material in gynogenetic offspring mainly relies on read-depth-dependent comparative analysis (RDC) and microsatellite markers. However, these methods suffer from high cost, operational complexity, low coverage, and low positive rates. This scheme provides a method for comprehensive and precise screening of genetic material derived from the paternal parent, yellow croaker, in the genome of gynogenetic large yellow croaker. This method can not only clarify the source of economic traits in gynogenetic offspring and develop specific molecular markers for gynogenetic large yellow croaker, but also contribute to variety identification and variety rights protection. This scheme will promote the genetic analysis of traits in gynogenetic large yellow croaker and the application of gynogenetic technology in breeding.

[0028] Based on the above, this solution also proposes an application of the identification method described above in one or more of the following:

[0029] 1) Develop a procedure for identifying heterologous paternal genetic material in the genomes of gynogenetic animals;

[0030] 2) Identification of heterologous paternal genetic material in the genomes of gynogenetic animals;

[0031] 3) Isolation and identification of parental genetic material in the genomes of hybrid animals;

[0032] 4) Develop molecular markers for identification of gynogenetic or hybrid animal breeds and protection of breed rights.

[0033] Based on the above, this scheme also proposes an identification system for the paternal yellow croaker DNA fragment in the genome of gynogenetic large yellow croaker, which applies the identification method described above.

[0034] Compared with existing technologies, the present invention, employing the above-described technical solution, has the following advantages: This solution identifies exogenous DNA in the genome of gynogenetic offspring by identifying paternally specific variant sites, analyzing the genotype of the offspring genome at paternally specific variant sites, performing multiple sequence alignment, and calculating the number and proportion of positive sites in the aligned blocks. Compared with traditional RDC methods, this solution is less costly because it does not require gynogenetic offspring genome sequences, and the required next-generation sequencing depth for both parents and offspring is only 10× or higher. Furthermore, the validation results of five candidate fragments using this solution show that all candidate fragments are paternal DNA fragments. Therefore, this solution has a higher positive rate. Moreover, compared with microsatellite methods, this solution utilizes almost all variants covering the entire reference genome, including SNPs, insertions, and deletions, to identify exogenous DNA in the genome of gynogenetic offspring. It can comprehensively analyze the integration of exogenous DNA fragments across the entire genome. Moreover, this solution mainly relies on bioinformatics analysis, which is not only cost-effective but also more time- and labor-saving. This solution provides technical support for the development of molecular markers for gynogenetic animals, breed identification, and breed rights protection. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 An example of a method for identifying paternal DNA fragments in the genome of gynogenetic large yellow croaker; Figure 1The sequences in the figure are hypothetical sequences, intended to illustrate the paternal DNA fragment identification method. They include: S1: Identification of the Specific Alter (SALT) site in yellow croaker; S2: Genotyping of gynogenetic large yellow croaker at the yellow croaker-specific mutation site. The figure shows the genotyping results of fragments 1 and 2. Fragment 1 has three positive sites, while fragment 2 has only one; S3: Obtaining the variant sequence by replacing the corresponding bases of the candidate fragment with the yellow croaker-specific variant bases; S4: Alignment of the variant sequence with the yellow croaker genome; S5: Multiple sequence alignment among the yellow croaker sequence, variant sequence, and candidate fragment sequence; further filtering of candidate fragments by comparing the number and percentage of positive sites within the alignment blocks; In the legend, fragment 1 has 3 positive sites in its block, representing 50% of the total, meeting the screening criteria and thus retained as a candidate fragment; fragment 2 has only 1 positive site in its block, representing only 1 / 6 of the total yellow croaker positive sites, failing the screening criteria and thus discarded.

[0037] Figure 2 This study validated the sequencing of candidate fragments derived from yellow croaker in the genome of the gynogenetic large yellow croaker gyno1 individual.

[0038] Figure 3 This study validated the sequencing of candidate fragments derived from yellow croaker in the genome of the gynogenetic large yellow croaker gyno2 individual.

[0039] Figure 4 To detect the candidate fragment NC_040011.1:18197014-18198736 of gynogenesis in large yellow croaker using SCAR markers, where NC_040011.1 represents the chromosome number of large yellow croaker and 18197014-18198736 represents the start and end sites of the fragment on the chromosome.

[0040] Figure 5 To detect the candidate fragment NC_040011.1:297860-302216 of gynogenesis in large yellow croaker using SCAR markers, where NC_040011.1 represents the chromosome number of large yellow croaker and 297860-302216 represents the start and end sites of the fragment on the chromosome. Detailed Implementation

[0041] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be particularly noted that the following embodiments are for illustrative purposes only and do not limit the scope of the invention. Similarly, the following embodiments are only some, not all, embodiments of the present invention, and all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] This embodiment describes a method for identifying paternal *Scoidea spp.* DNA fragments in the genome of gynogenetic large yellow croaker (C. croaker), comprising: preparing genome sequences of large yellow croaker and *Scoidea spp.*, as well as second-generation resequencing data of common large yellow croaker, gynogenetic large yellow croaker, and *Scoidea spp.*; then performing quality control processing on the second-generation resequencing data; then comparing all second-generation resequencing data with the large yellow croaker genome sequence one by one; then marking repetitive sequences and identifying *Scoidea spp.*-specific variant sites; and finally performing targeted genotyping of gynogenetic large yellow croaker at the *Scoidea spp.*-specific variant sites, targeting the gynogenetic large yellow croaker towards the gynogenetic genome. Yellow croaker-specific variation information was added to the genotyping file of gynogenetic large yellow croaker. Then, paternal positive sites in the genome of gynogenetic large yellow croaker were screened, and positive sites were merged into continuous candidate fragments. Subsequently, the bases in the positive sites of the candidate fragments were replaced with yellow croaker-specific variant bases to obtain variant sequences. The variant sequences were then compared with the yellow croaker genome. Candidate fragments were filtered by multiple sequence alignment based on the number and proportion of positive sites in the alignment blocks. Finally, the yellow croaker fragments in the genome of gynogenetic large yellow croaker were sequenced for verification and SCAR marker detection.

[0043] in, Figure 1 This paper illustrates an example of the method for identifying paternal DNA fragments in the genome of gynogenetic large yellow croaker in this scheme.

[0044] Specifically, the solution in this embodiment includes the following steps:

[0045] Step 1. Data Preparation

[0046] S1) Preparation of genome sequence files

[0047] Download the genome data files of large yellow croaker and yellow croaker in FASTA format from the NCBI database, and rename them to LC.fa and NA.fa, respectively.

[0048] S2) Preparation of second-generation resequencing data

[0049] Download Illumina PE150 resequencing data from a population of 10 large yellow croakers and 10 yellow croakers from the NCBI database. The resequencing data must have an average sequencing depth of 10× or higher, be paired-end sequencing, and be in FASTQ format. The accession numbers for the large yellow croaker resequencing data are: SRR11658747, SRR11658746, SRR11658745, SRR11658744, SRR11658743, SRR11658742, SRR11658741, SRR11658740, SRR11658739, and SRR11658737. The accession numbers for the resequencing data of *Cinnamomum campestris* are: SRR21161137, SRR21161139, SRR21161140, SRR21161142, SRR21161143, SRR21161146, SRR21161151, SRR21161155, SRR21161161, and SRR21161172. The large yellow croaker samples are designated lc1-lc10, and the *Cinnamomum campestris* samples are designated na1-na10. Simultaneously, resequencing was performed on 10 gynogenetic large yellow croakers using an Illumina PE150 scanner, with an average sequencing depth of over 10×; these samples are designated gyno1-gyno10. The sequencing data files for each sample are named as sample_R1.fastq and sample_R2.fastq, for example, the sequencing data for lc1 are lc1_R1.fastq and lc1_R2.fastq.

[0050] Step 2. Quality control of next-generation sequencing data

[0051] Quality control is performed on all second-generation data, including:

[0052] S1) Save the sample names of all samples to samples.txt, the code of which includes the following:

[0053] ls*_R1.fastq|sed's / _R1.fastq / / '>samples.txt

[0054] S2) Remove low-quality reads and sequences; the code includes the following:

[0055]

[0056] The above procedure removes reads with Q < 20, N > 10%, and length < 75 bp, thus removing polyG / polyX sequences. Here, {1} represents a placeholder and indicates a sample name in the command (the same meaning applies below, so it will not be repeated).

[0057] Step 3. Sequence alignment

[0058] S1) Compare all the quality-controlled resequencing data with the large yellow croaker genome one by one. The code includes the following:

[0059] bwa mem-t 8LC.fa{1}_clean_R1.fastq{1}_clean_R2.fastq>{1}.sam

[0060] S2) Compare file formats and remove low-quality comparisons. The code includes the following:

[0061] samtools view-bq 30{1}.sam>{1}.bam

[0062] S3) Compare and sort the files, the code of which includes the following:

[0063] samtools sort-@4{1}.bam-o{1}.sorted.bam

[0064] Step 4: Mark repeating sequences

[0065] S1) Create the index file, the code of which includes the following:

[0066] samtools index{1}.sorted.bam

[0067] S2) Marks repeating sequences, and its code includes the following:

[0068] nohup parallel-j 4"gatk MarkDuplicates-I{1}.sorted.bam-O{1}.markdup.bam-M{1}.markdup_metrics.txt--REMOVE_DUPLICATES false2>>MarkDuplicates.log"::::samples.txt>>MarkDuplicates.log 2>&1&

[0069] Step 5. Identification of specific variant sites in yellow croaker

[0070] S1) Rebuild the index file, the code of which includes the following:

[0071] nohup parallel samtools index{}:::*.markdup.bam 2>markdup.index.log&

[0072] S2) Add @RG information to the alignment file, the code of which includes the following:

[0073] parallel-j 4"picard AddOrReplaceReadGroups I={1}.markdup.bamO={1}.markdup.fixed.bam RGID={1}.RG1 RGLB=lib1 RGPL=illuminaRGPU=unit1 RGSM={1}VALIDATION_STRINGENCY=SILENT"::::samples.txt

[0074] S3) Indexes the repaired alignment files, and its code includes the following:

[0075] parallel-j 4"samtools index{1}.markdup.fixed.bam"::::samples.txt

[0076] S4) Individual variation detection of single samples of common large yellow croaker and yellow croaker, the code includes the following:

[0077] nohup"gatk--java-options'-Xmx4G'HaplotypeCaller-R LC.fa-I{1}.markdup.fixed.bam-O{1}.g.vcf.gz-ERC GVCF--sample-name{1}-GStandardAnnotation-G AS_StandardAnnotation 2>>{1}.HaplotypeCaller.error.log"::::sample2.txt>>HaplotypeCaller.log 2>&1&

[0078] The sample2.txt file contains the sample names of common yellow croaker and yellow croaker, with each sample on a separate line.

[0079] S5) Yellow croaker population variation detection, the code includes the following:

[0080] gatk CombineGVCFs-R LC.fa-V na1.g.vcf.gz-V na2.gv cf.gz-Vna3.g.vcf.gz-V na4.g.vcf.gz-V na5.g.vcf.gz-V na6.g.vcf.gz-V na7.g.vcf.gz-Vna8.g.vcf.gz-V na9.g.vcf.gz-V na10.g.vcf.gz-O na_combined.g.vcf

[0081] gatk GenotypeGVCFs-R LC.fa-V na_combined.g.vcf.gz-Ona_genotyped.vcf.gz

[0082] S6) Preliminary screening of yellow croaker-specific loci (compared to the large yellow croaker reference genome), the codes of which include the following:

[0083] bcftools view-i'AF[0]=1.00&&AN>=14&&QUAL>30'na_genotyped.vcf.gz-Oz-o na_fixed.vcf.gz

[0084] S7) Population variation detection for common large yellow croaker, the codes of which include the following:

[0085] gatk CombineGVCFs-R LC.fa-V lc1.g.vcf.gz-V lc2.gv cf.gz-V lc3.g.vcf.gz-V lc4.g.vcf.gz-V lc5.g.vcf.gz-V lc6.g.vcf.gz-V lc7.g.vcf.gz-Vlc8.g.vcf.gz-Vlc9.g.vcf.gz-V lc10.g.vcf.gz-O lc_combined.g.vcf

[0086] gatk GenotypeGVCFs-R LC.fa-V lc_combined.g.vcf.gz-Olc_genotyped.vcf.gz

[0087] S8) Further screening for specific variant sites in yellow croaker (compared to the common large yellow croaker population), the codes of which include the following:

[0088] gatk IndexFeatureFile-I na_fixed.vcf.gz-O na_fixed.vcf.gz.tbi

[0089] gatk IndexFeatureFile -I lc_genotyped.vcf.gz -O lc_genotyped.vcf.gz.tbi

[0090] gatk SelectVariants -R LC.fa -V na_fixed.vcf.gz --discordancelc_genotyped.vcf.gz -O na_specific.vcf

[0091] S9) Only biallelic loci are retained to obtain the final yellow croaker-specific mutation loci, the codes of which include the following:

[0092] bcftools view-m2-M2 na_specific.vcf -O zo na_specific_biallelic.vcf.gz

[0093] S10) Add a unique ID to the na_specific_biallelic.vcf.gz file to facilitate subsequent matching. The code includes the following:

[0094] bcftools annotate --set-id'%CHROM\_%POS'na_specific_biallelic.vcf.gz -O zo na_specific_id.vcf.gz

[0095] S11) Create an index for the na_specific_id.vcf.gz file, the code of which includes the following:

[0096] bcftools index-ft na_specific_id.vcf.gz

[0097] Step 6. Targeted genotyping of gynogenetic large yellow croaker at specific variant sites in yellow croaker

[0098] To reduce the amount of data computation, targeted genotyping was performed on gynogenetic large yellow croaker at specific mutation sites. The analysis process is illustrated below using the gynogenetic individual gyno1 as an example.

[0099] S1) Genotypes of gynogenetic large yellow croakers at specific mutation sites in yellow croakers were detected one by one and a .g.vcf file was generated, the code of which includes the following:

[0100] gatk HaplotypeCaller-R LC.fa-I gyno1.markdup.fixed.bam-Ogyno1.specific.g.vcf.gz-ERC GVCF--sample-name gyno1-L na_specific_id.vcf.gz--active-probability-threshold 0.001--minimum-mapping-quality 20

[0101] S2) Convert the .g.vcf files of each sample into a standard .vcf file. The code includes the following:

[0102] gatk GenotypeGVCFs-R LC.fa-V gyno1.specific.g.vcf.gz-Ogyno1.specific.vcf.gz

[0103] Step 7. Add yellow croaker-specific variation information to the gynogenetic large yellow croaker genotyping file gyno1.specific.vcf.gz.

[0104] S1) Add the "Specific Alter (SALT)" field to the annotation file na_specific_id.vcf.gz. The code includes the following:

[0105] # Add a SALT field to the INFO column of the data row in na_specific_id.vcf.gz

[0106] bcftools view na_specific_id.vcf.gz|\awk'BEGIN{OFS="\t"} / ^# / {print; next}{$8=$8";SALT="$5;print}'|\bcftools view-Oz-ona_specific_id_with_SALT.vcf.gz

[0107] gunzip -c na_specific_id_with_SALT.vcf.gz>temp.vcf

[0108] vi temp.vcf

[0109] #Insert the following code on the line before the first #CHROM line in the temp.vcf file:

[0110] ##INFO=<ID=SALT,Number=1,Type=String,Description="Alternatealleleannotation">

[0111] #Recompress and rebuild the index, the code includes the following:

[0112] bgzip -c temp.vcf>na_specific_id_with_SALT.vcf.gz

[0113] tabix -p vcf na_specific_id_with_SALT.vcf.gz

[0114] S2) Add the yellow croaker-specific variant marker to the gyno1.specific.vcf.gz file of the gynogenetic large yellow croaker individual. The code includes the following:

[0115] #Add SALT field to VCF header

[0116] echo'##INFO=<ID=SALT,Number=1,Type=String,Description="SpecificALT from nibe"> salt_header.txt

[0117] # Mark the yellow croaker-specific variants into the gynogenetic individual's gyno1.specific.vcf.gz file

[0118] bcftools annotate-a na_specific_id_with_SALT.vcf.gz-c+SALT--header-lines salt_header.txt gyno1.specific.vcf.gz-O z-ogyno1.specific.marked.vcf.gz

[0119] Step 8: Screening for paternal positive loci in the genome of gynogenetic large yellow croaker.

[0120] This scheme considers loci that simultaneously meet the following conditions as paternal positive loci:

[0121] 1) The genotype of the gynogenetic large yellow croaker at the specific mutation site of the yellow croaker is heterozygous (0 / 1) or homozygous variant (1 / 1);

[0122] 2) The alleles of gynogenesis in large yellow croaker at the specific mutation site of yellow croaker are the same as those of yellow croaker.

[0123] 3) The total depth DP is required to be ≥5, and for heterozygotes, the depth of variant alleles is required to be ≥2.

[0124] For homozygous variants, the reference allele depth must be 0. The specific steps include:

[0125] S1) Basic filtering, the code of which includes the following:

[0126] bcftools view-i'QUAL>=20&&FORMAT / DP>=5&&(GT="0 / 1"||GT="1 / 1")&&ALT==SALT'gyno1.specific.marked.vcf.gz-Oz-ogyno1.temp.vcf.gz

[0127] S2) Precise filtering of AD values, the code includes the following:

[0128] bcftools query-f'%CHROM\t%POS\t%REF\t%ALT\t[%GT\t%AD\n]'gyno1.temp.vcf.gz|awk-F'\t ” BEGIN{OFS="\t"}{split($6,ad,",");if(($5=="0 / 1"&&ad[2]>=2)||($5=="1 / 1"&&ad[1]==0))print$1,$2,$3,$4}'>gyno1.positions.txt

[0129] S3) Extract the final result, the code of which includes the following:

[0130] bcftools view-T gyno1.positions.txt gyno1.temp.vcf.gz-Oz-ogyno1.true_positives.vcf.gz

[0131] rm gyno1.temp.vcf.gz gyno1.positions.txt

[0132] Example 9. Combined positive sites are continuous candidate fragments.

[0133] Based on the location information of positive sites in the gynogenesis genome of large yellow croaker within the reference genome coordinate system, adjacent positive sites were merged into continuous candidate fragments (maximum gap 1 kb), while short fragments (>1 kb) were filtered out. The specific process is as follows:

[0134] S1) Convert gyno1.true_positives.vcf.gz from .vcf to BED format. The code includes the following:

[0135] bcftools query-f'%CHROM\t%POS0\t%POS\t%ID\n'gyno1.true_positives.vcf.gz>gyno1.positives.bed

[0136] S2) Merges adjacent sites (maximum gap 1kb), and its code includes the following:

[0137] bedtools merge-i gyno1.positives.bed -d 1000>gyno1.fragments.bed

[0138] S3) Filters short segments (>1kb), and its code includes the following:

[0139] awk'$3-$2>=1000'gyno1.fragments.bed>gyno1.final_fragments.bed

[0140] Step 10. Obtaining the mutant sequence

[0141] The mutant sequence was obtained by replacing the bases at positive sites of candidate fragments with bases specifically mutated by yellow croaker. The specific process is as follows:

[0142] Add an ID column to the S1)gyno1.final_fragments.bed file, with the following code:

[0143] awk'BEGIN{FS=OFS="\t";print"ID","Chromosome","Start","End"}{print$1":"$2"-"$3,$0}'gyno1.final_fragments.bed>gyno1.final_fragments-V2.bed

[0144] S2) Create the index, the code of which includes the following:

[0145] bcftools index-c gyno1.specific.marked.vcf.gz

[0146] S3) Introduce the yellow croaker-specific variant into the candidate fragment reference sequence, the code of which includes the following:

[0147] tail-n+2gyno1.final_fragments-V2.bed|cut-f1|while read-r region; dohr=${region%:*}; start_end=${region#*:}; start=${start_end%-*}; end=${start_end#*-}; {bcftools consensus-s"gyno1"-f<(samtools faidx / disks / node2_60T / zhouzhaolong / SGM / LC.fa"$region")-H A-onibe_"${chr}_${start}_${end}".fagyno1.specific.marked.vcf.gz 2>>error.log&&sed-i"s / >.* / >${region} / "nibe_"${chr}_${start}_${end}".fa;}||echo "Failed on$region">>error.log;done

[0148] cat nibe_*.fa>gyno1_nibe_specific_fragments.fa

[0149] S4) Remove newline characters within the mutated sequence. The code includes the following:

[0150] awk' / ^> / {printf("\n%s\n",$0);next;}{printf("%s",$0);}END{printf("\n");}'gyno1_nibe_specific_fragments.fa|tail-n+2>gyno1_nibe_specific_fragments-V2.fa

[0151] Step 11. Alignment of candidate fragment variant sequences with the yellow croaker genome.

[0152] S1) The genome library of the yellow croaker was constructed, and the code includes the following:

[0153] makeblastdb-in NA.fa-dbtype nucl-parse_seqids-out NA_db

[0154] S2) The candidate fragment variant sequence is aligned to the yellow croaker genome using blastn, and the code includes the following:

[0155] blastn-query gyno1_nibe_specific_fragments-V2.fa-db NA_db-outfmt 6-outgyno1_blast_results.txt

[0156] Step 12. Filter candidate fragments using multiple sequence alignment and the proportion of yellow croaker-specific variations within the alignment block.

[0157] Each blastn hit is used as an alignment block. The reference sequence for *Cyprinus maculatus* (Sequence 1), the variant sequence of the candidate fragment (Sequence 2, where the positive site has been replaced with *Cyprinus maculatus*-specific variant bases), and the reference sequence for the large yellow croaker candidate fragment (Sequence 3, where the positive site has not been replaced with *Cyprinus maculatus*-specific variant bases) are extracted from each block. Multiple sequence alignment of these three sequences is performed using MAFFT. If at a variant site, Sequence 1 and Sequence 2 have the same bases but are different from Sequence 3, then that site is a positive site. If at a variant site, Sequence 2 and Sequence 3 have the same bases but are different from Sequence 1, then that site is a non-positive site. The percentage of positive sites within a block is calculated as the number of positive sites / (number of positive sites + number of non-positive sites). Candidate fragments with a positive site percentage < 30% or ≤ 2 positive sites within a block are excluded.

[0158] S1) Analysis Plan Development

[0159] This solution details the analysis process in a Python script named gyno1_MSA.py, the script content of which is as follows:

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171] S2) Run the script, the code of which includes the following:

[0172] python3 gyno1_MSA.py

[0173] Interpretation of S3 script execution results

[0174] After the script runs, it generates a file named gyno1_result.xls, which contains 10 columns: Fragment_ID (candidate fragment ID, indicating the position of the candidate fragment in the large yellow croaker genome), Fish_Block_Position (candidate fragment block ID, indicating the position of the block in the large yellow croaker genome), Nibe_Block_Position (the position of the block in the large yellow croaker genome), Positive_Sites (number of positive sites in the block), Non_Positive_Sites (number of positive sites in the block), Proportion (proportion of positive sites in the alignment block), Block_Length (block length), Nibe_Sequence (large yellow croaker sequence in the block), Replaced_Fish_Sequence (variant sequences in the block where the bases at the positive sites of the candidate fragment have been replaced by large yellow croaker-specific variants), and Unaltered_Fish_Sequence (reference sequences in the block where the bases at the positive sites of the candidate fragment have not been replaced by large yellow croaker-specific variants).

[0175] Step 13. Sequencing verification and SCAR marker detection of the yellow croaker fragment in the genome of gynogenetic large yellow croaker.

[0176] S1) Sequencing validation based on universal primers

[0177] Universal primers were designed for conserved regions in the sequences of large yellow croaker and yellow croaker. PCR amplification was performed using the genomes of common large yellow croaker, gynogenetic large yellow croaker, and yellow croaker as templates. The amplified products were sequenced, and candidate fragments were verified by multiple sequence alignment. In this case, two candidate fragments were identified for gynogenetic individual 1 (gyno1) and gynogenetic individual 2 (gyno2), and a pair of universal primers was designed for each. PCR amplification was performed using common large yellow croaker, gynogenetic large yellow croaker, and yellow croaker as templates, and the PCR products were sequenced. The sequencing results were compared. The results showed that both gyno1 and gyno2 contained two fragments, one from large yellow croaker and the other from yellow croaker (see reference). Figure 2 , Figure 3 This result proves that the selected candidate fragments did indeed come from yellow croaker.

[0178] S2) PCR amplification and verification based on yellow croaker-specific primers

[0179] Two candidate fragments, fragment 1 and fragment 2, distributed in two different gynogenetic large yellow croaker genomes, were targeted. Specific primers for large yellow croaker were designed at the specific variant sites in these fragments. PCR amplification was performed using genomes of common large yellow croaker, gynogenetic large yellow croaker, and large yellow croaker as templates to detect the SCAR marker in gynogenetic large yellow croaker. Agarose gel electrophoresis results showed that bands were only present in the corresponding gynogenetic individuals and large yellow croaker (reference). Figure 4 , Figure 5 This result further confirms the candidate fragments, and these candidate fragments can be used for the development of molecular markers for gynogenesis in large yellow croaker.

[0180] The above description is only a part of the embodiments of the present invention and does not limit the scope of protection of the present invention. Any equivalent device or equivalent process transformation made based on the content of the present invention specification and drawings, or direct or indirect application in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for identifying a DNA fragment of male O. marmoratus in the genome of gynogenetic P. major, characterized in that, It comprises: By identifying the genotype of the gynogenetic P. olivaceus at the specific mutation site of N. eurystoma, screening the positive site from the male N. eurystoma, then detecting the DNA fragment from the male N. eurystoma by multi-sequence alignment and calculating the number and proportion of positive sites in the alignment block, and finally confirming by sequencing or SCAR marker detection technology.

2. The method for identifying the DNA fragment of the male O. joniai in the genome of the gynogenetic P. olivaceus according to claim 1, characterized in that, It comprises: Preparation of P. olivaceus and N. eurystoma genome sequences, and second-generation resequencing data of P. olivaceus, gynogenetic P. olivaceus and N. eurystoma; then quality control processing of the second-generation resequencing data, alignment of all second-generation resequencing data with P. olivaceus genome sequence, labeling of repetitive sequences and identification of N. eurystoma specific mutation sites, then targeted genotyping of gynogenetic P. olivaceus at the N. eurystoma specific mutation sites, adding N. eurystoma specific mutation information to the gynogenetic P. olivaceus genotyping file; then screening the male positive sites in the gynogenetic P. olivaceus genome, merging the positive sites into continuous candidate fragments, then replacing the bases in the positive sites of the candidate fragments with N. eurystoma specific mutation bases to obtain mutation sequences, aligning the mutation sequences with the N. eurystoma genome, filtering the candidate fragments by multi-sequence alignment and according to the number and proportion of positive sites in the alignment block, and finally sequencing verification and SCAR marker detection of the N. eurystoma fragments in the gynogenetic P. olivaceus genome.

3. Use of the method of identification according to claim 1 or 2 for one or more of the following, characterized in that, It comprises: 1) Development of a program for identifying heterologous paternal genetic material in the genome of gynogenetic animals; 2) Identification of heterologous paternal genetic material in the genome of gynogenetic animals; 3) Separation and identification of paternal and maternal genetic material in the genome of hybrid animals; 4) Development of molecular markers for the identification of gynogenetic or hybrid animal breeds and breed protection.

4. An identification system of a DNA fragment of male O. marmoratus in the genome of gynogenetic P. major, characterized in that, The application of the identification method of claim 1 or 2.

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